EPA-560/2-76-005
A LITERATURE SURVEY ORIENTED TOWARDS ADVERSE
ENVIRONMENTAL EFFECTS RESULTANT FROM THE USE
OF AZO COMPOUNDS, BROMINATED HYDROCARBONS
EDTA, FORMALDEHYDE RESINS, AND 0-
NITROCHLOROBENZENE
FINAL REPORT
ENVIRONMENTAL PROTECTION AGENCY
OFFlCE^JOXIC^UBStANCES
WASHlNGf6N,~D^c720460"
-------
EPA-560/2-76-005
A LITERATURE SURVEY ORIENTED TOWARDS ADVERSE
ENVIRONMENTAL EFFECTS RESULTANT FROM THE USE
OF AZO COMPOUNDS, BROMINATED HYDROCARBONS,
EDTA, FORMALDEHYDE RESINS, AND 0-NITROCHLOROBENZENE
Contract No. 68-01-2212
Project Officer
Frank D. Kover
Office of Toxic Substances
Environmental Protection Agency
Washington, D.C. 20460
Prepared for
Environmental Protection Agency
Office of Toxic Substances
Washington, D.C. 20460
June 1976
-------
TABLE OF CONTENTS
Summary and Conclusion
Azo Compounds 1
Bromlnated Hydrocarbons 276
EDTA 372
Formaldehyde Resins 425
o-Nitrochlorobenzene 470
I, Physical Properties
Azo Compounds 3
Brotninated Hydrocarbons 278
EDTA 373
Formaldehyde Resins 429
o-Nitrochlorobenzene 471
II. Production
Azo Compounds 4
Brominated Hydrocarbons 280
EDTA 381
Formaldehyde Resins 432
o-Nitrochlorobenzene 471
III. Use
Azo Compounds 30
Brominated Hydrocarbons 282
EDTA 381
Formaldehyde Resins 436
o-Nitrochlorobenzene 472
li
-------
TV. Current Practice
Azo Compounds 39
Brominated Hydrocarbons 284
EDTA 385
Formaldehyde Resins 441
o-Nitrochlorobenzene 472
V. Environmental Contamination
Azo Compounds -„
Brominated Hydrocarbons 285
EDTA 385
Formaldehyde Resins 442
o-Nitrochlorobenzene 472
VI. Monitoring and Analysis
Azo Compounds 44
Brominated Hydrocarbons 286
EDTA 385
Formaldehyde Resins 444
o-Nitrochlorobenzene 472
VII. Chemical Reactivity
Azo Compounds 52
Brominated Hydrocarbons 292
EDTA 389
Formaldehyde Resins 451
o-Nitrochlorobenzene 473
ill
-------
VIII. biology
Azo Compounds 66
Brominated Hydrocarbons 297
EDTA 391
Formaldehyde Resins 452
o-Nitrochlorobenzene 473
IX. Environmental Effects
A. Persistence and/or degradation
Azo Compounds 102
Brominated Hydrocarbons 315
EDTA 401
Formaldehyde Resins 454
o-Nitrochlorobenzene 474
B. Environmental Transport
Azo Compounds 103
Brominated Hydrocarbons 322
EDTA 402
Formaldehyde Resins 454
o-Nitrochlorobenzene 475
C. Bioaccumulation
Azo Compounds 103
Brominated Hydrocarbons 322
EDTA 402
Formaldehyde Resins 454
o-Nitrochlorobenzene 475
iv
-------
X. Toxicity
Azo Compounds 103
Brominated Hydrocarbons 324
EDTA 402
Formaldehyde Resins 455
o-Nitrochlorobenzene 475
XI. Current Regulations
Azo Compounds 210
Brominated Hydrocarbons 355
EDTA 413
Formaldehyde Resins 461
o-Nitrochlorobenzene 477
XII. Standards
Azo Compounds 210
Brominated Hydrocarbons 360
EDTA 413
Formaldehyde Resins 463
o-Nitrochlorobenzene 477
Literature Cited
Azo Compounds 211
Brominated Hydrocarbons 361
EDTA 414
Formaldehyde Resins 466
o-Nitrochlorobenzene 478
Appendix
Azo Compounds 231
-------
AZO COMPOUNDS
SUMMARY AND CONCLUSION AS TO DEGREE OF HAZARD
A very large number of azo dyes is in production throughout the
world, but amounts made vary over a wide range and change considerably
from year to year according to the whims of fashion and design. Biologi-
cal studies do not seem to have been done on the majority of these.
Other than an occasional case of skin allergy to a clothing dye, there
doesn't seem to be any need for concern about the azo dyes used in
cloth, paints, plastics, inks, etc. The dyes used in food, drugs, and
cosmetics vary from one country to another, each of the latter seeming
to have a different set of standards for rating studies for toxicity in
laboratory animals. Currently undergoing investigation is the neglected
area of teratogenic effects. Because of the wide range of foods a
particular dye may be found in, it has proven rather difficult to extra-
polate "no adverse effect" levels in animals to humans and then set a
daily overall consumption limit.
Metabolism studies of the dyes have indicated that many are cleaved
only by the intestinal flora, a rather variable factor even in highly in-
bred populations of laboratory animals. Both oil and water soluble dyes
seem able to pass through the intestinal wall in both directions, thus
complicating these studies.
Teratogenetic studies have been done most frequently on Trypan Blue
and its related biological stains. Trypan Blue has been shown to be a
variably complex mixture, neither in part nor in whole consistently terato-
genic. The related dyes show a lesser degree of teratogenicity, the
studies complicated by mislabeling by manufacturers.
-------
Carcinogenicity of azo dyes in humans has not been demonstrated,
possible occupational cases being compromised by concurrent exposure
to carcinogenic starting materials. Many studies have been conducted
on derivatives of ring- and N-methylated 4-aminoazobenzene. Rats have
developed cancers of the liver when fed some of these for two months,
usually in a low-protein, low-riboflavine diet. Other laboratory animals
show much less to no susceptibility to these hepatocarcinogens. Researchers
have been unable to unravel the sequence of events leading to the ini-
tiation of tumor growth, there being no correlations of internal physi-
cal or chemical changes and relative carcinogenicities to guide them.
Studies on the flour additive azodicarbonamide have shown that it is
completely altered to another compound in the baking process, biurea,
which was not toxic at the level involved.
Considering the number of azo compounds in distribution and the uses
to which they are being put, the lack of hazard is remarkable. The
greatest danger appears to be from the ingestion of unauthorized food
dyes. Adequate supervision of imports and thorough testing of new dyes
should protect the consumer.
-------
AZO COMPOUNDS
I. PROPERTIES
This report is confined to compounds having the linkage C-N=N-C.
No attempt was made to compile a list of melting points for the azo dyes
or the azo compounds used in cancer research because: most commercial
products, even dyes authorized for human consumption, are mixtures of
different compounds or isomers; structural features such as sulfonic acid
salts and great complexity tend to induce decomposition prior to melting;
the heating process could induce changes in the isomerism about the azo
linkage(s), or tautomerism about highly electropositive/electronegative
functional groups prior to melting, so that any melting point might not
be for the original structure. In general the azo dyes are not water
soluble unless they have at least one sulfonic acid group (-S03H), and
too many of these makes the compound insoluble in organic solvents. All
of the food, drug, and cosmetic dyes are water soluble, but nearly insol-
uble in most organics. Of the FD&C's, Red No. 2 has a solubility in gly-
cerine or propylene glycol 1.5 or 0.083 times, respectively, that in water;
for Red No. 4 the comparable figures are 0.55 or 0.16; for Red No. 40,
0.14 or 0.07; for Yellow No. 5, 1.6 or 0.68; for Yellow No. 6, 0.63 or 0.11.
Azobenzene, the simplest azoaromatic, exists at room temperature in
the trans form, mp 68°C, but can be isolated under colder conditions in
the cis form, mp 71°C. The trans form has a specific gravity of 1.203
(20/4°), a vapor pressure of 1 mm Hg at 103.5°C, a bp of 300°C, and is
soluble in organics.
2,2'-Azobisisobutyronitrile, NC C(CH3)2N = NC (CH3)2CN, melts at
105°C with decomposition, is soluble in organics, insoluble in water.
-------
Azodicarbonamide, H2NC(0)N = NC(0)NH2, melts at 180-4°C with decom-
position, is soluble in dimethyl sulfoxide, insoluble in the common or-
ganics and in water.
Sawicki (1957) studied the tautomerism of 73 aminoazobenzerie com-
pounds in a 50% alcoholic HC1 solution by measuring the UV spectra, re-
porting values for pK and for the ratio of the intensities of the ab-
3.
sorptions due to the proton being on one of the azo N's or on the amino N.
Gerson and Heilbronner (1962) did a similar study on p-N,N-
dimethylaminoazobenzene and ten derivatives, reporting the values for
mp, difference in pK between the parent and the derivative, and the
3.
absorption intensity ratio. They also studied the tautomerism of p,p'-
bis(dimethylamino)azobenzene in a variety of acid-solvent systems
(pp. 51-9 of the indicated reference).
Bershtein and Ginzburg (1972) have reviewed the literature through
1969 concerning the tautomerism of hydroxy- and aminoazo compounds.
In Section VI (Monitoring and Analysis) of this report are reviewed
a number of papers which discuss the nature of the impurities in speci-
fic azo compounds.
II. PRODUCTION
Production of azo dyes and azo pigments in the United States has
been increasing steadily since 1958 following a plateau from 1952-1958.
Statistics for the dyes and pigments are given separately in the following
tables.
-------
Table 1. United States Production of Azo Dyes
1952
1953
1954
1955
1956
1957
1958
1959
1960
1961
1962
22. 3*
25.9
24.1
27.3
23.2
23.2
18.2
23.6
20
20.5
23.6
1963
1964
1965
1966
1967
1968
1969
1970
1971
1972
24.6
25,9
30.4
31.8
26.8
32,3
34.1
37.3
41.8
a Units are in 1,000 metric tons
Table 2. United States Production of Azo Pigments
1961
1962
1963
1964
1965
1966
8,82a
9.08
9.45
10.0
10.9
11.5
1967
1968
1969
1970
1971
12.2
12,5
14.1
12.9
13.9
a Units are in 1,000 metric tons
Production statistics on individual azo dyes and pigments for 1971
and 1972 (including imports for 1972) follow. As with the overall annual
figures these have been taken from the United States Tariff Commission
Reports. The dyes for which no figures were given therein (to protect the
-------
few companies making them) are included in an overall compilation indicat-
ing the manufacturers. Many azo dyes have, very likely, been excluded
because it was not possible to identify them as such, the available
volumes of the only reference work Colour Index not covering the latest
ten years.
Table 3. Production (1971, 1972) and Importation (1972) in
the United States of Individual Azo Dyes and Pigments
Yellow
9
11
17
19
23
25
34
36
38
40
42
44
54
64
65
70
72
76
99
121
Aoid Dyes3'
b
34.6
258
-
142
-
35.4
87.2
72.6
154
36.8
7.72
17.3
-
N.R.
-
-
23.6
47.7
_
c
24.1
214
-
127
-
N.R.
74.5
-
45.4
40
N.R.
50
-
35.4
-
-
17.3
21.4
_
0.91d
-
-
76
5.1
10.7
-
1.95
14.7
-
2.4
-
-
8.75
-
0.30
0.70
-
0.25
0.45
-------
Orange
Red
124
127
128
135
136
151
159
7
8
10
19
24
28
33
51
60
61
64
74
92
94
102
1
4
6
14
N.R.
1.50
1.02
58.5
0.50
591 600
267 213
254 224 0.20
128.5 172 0.25
123 131
2.23
425 332
3.41
5.16
1.25
86.3 110
0.50
21.8
37.2 40.4
0.68
11.8
0.90
197 173
46.3 51.3
0.02:
51.3 N.R.
-------
18
26
32
35
37
42
57
73
85
88
89
99
111
114
115
127
131
134
137
151
155
157
158
161
179
182
186
52.2 48.1
20.4 17.3
0.91
0.11
35.4 38.6 0.70
3.55
20.3
117 107 1.61
73.6 57.2 0.68
372 532
8.2 19.1
79 76.8 0.25
32.2
156 192 4.10
34.1 17.7
5.25
11.1
0.95
81.7 90.7
292 446 0.27
0.35
1.61
0.455
3.41
0.60
30.4 32.7
12.3
-------
Violet
Blue
240
249
251
252
257
258
259
260
261
263
266
274
282
283
1
3
5
7
12
14
90
92
113
118
120
151
154
0 . 10
6.31
5.91
3.23
10.9
1.90
1.31
12 . 1
1.05
3.76
55.9 122 22.8
1.25
1.80
2.25
17.3 13.2
39.6 55.9
0.451
43.6 61.8
_
1.80
0.125
44.5 N.R.
378 382 45.4
40.4 33.6
15.4 0.371
1.72
0.60
-------
Gfeen
Black
156
158/158A 45.9
163
183
184
187
193
199
205
20 23.6
60
68
14 394
33
83
85
127
224
226
227
235
239
253
264
1 427
24
2.16
55
0.05
0.326
1.24
5.06
0.60
0.125
2.75
27.7
0.20
2.18
338
L8.1
8.74
5.50
5.00
5.52
0.266
0.651
22.8
24.6
0.114
4.67
474 2.36
24.5 1.28
10
-------
Azoi-G Diazo
Component 4, base
Orange
Red
Brown
52 315 442
76
82
84
107 93.5 145
108
117
128
131
132
139
81.3 89
Basic Dyes
1 139 N.R.
2 224 203
28
29
30
18
23
24
25
1 29.5 66.8
4 218 218
-
1.50
0.150
0.97
14.1
0.60
3.00
0.80
19.6
19.1
7.61
-
-
0.274
1.82
0.227
8.26
0.227
38.4
0.341
1.93
-
0.05
-------
Orange
4
8
12
27
28
29
44
50
69
84
93
95
98
109
110
1
8
26
29
34
37
39
66
72
73
81
Dire at Dyes
214
-
117
_
110
19.5
396
221
-
346
-
-
-
-
-
10
28.2
26,8
56.7
49.1
17.7
88.6
_
180
_
N.R.
226
-
91
-
108
14.5
506
229
-
328
-
-
-
-
-
N.E.
49.5
31.8
46.4
50.9
11.8
104
_
148
51.4
36.4
_
0.65
0.15
2.01
-
-
1.82
_
0.227
_
1.40
0.85
22.5
0.57
1.18
-
-
-
-
-
-
-
1.24
-
-
_
-------
Red
102
106
107:
1
2
3
4
9
10
11
13
16
23
24
26
28
31
37
39
62
72
75
79
80
81
83
84
117
-
-
56.4
113
_
18.6
-
4.55
-
20.4
N.R.
109
181
43.2
48.1
7.27
49.1
67.3
-
85.9
9.1
37.2
274
243
106
_
189
-
-
58.1
92
-
30
-
N.R.
-
N.R.
60.5
111
164
47.7
79
_
48.6
73.6
-
123
7.72
102
305
242
114
_
-
1.75
17.5
5.16
_
1.36
-
4.91
-
0.60
-
-
0.50
-
-
2.27
-
-
-
2.04
-
1.68
3.03
1.02
-
3.00
0.226
13
-------
Violet
Blue
89
122
123
152
173
205
207
212
218
7
9
47
48
51
93
95
1
2
6
8
10
15
22
24
25
67
2.94
N.R.
N.R.
3.64
1.00
1.80
4.08
0.795
0.91
N.R. 2.66
87.6 68.6
13.7
9.03
10.9 1.93
9 . 10
2.50
136 172
580 466
119 131
64 106
3.41
94 108
10.45
N.R.
22.7 29.5 0.455
N.R.
Ill
-------
Green
71
76 53,2
78 64
80 286
98 39
112
120/120A
126 92
149
156
158
207
211
218 479
225
239
1 109
6 235
26
33
51
59
67
68
69
74
53.6
30.9
60.4
256
154
0
66.3 30
65.5
2
2
24
— s
2
499
_ o
0
106
184
2
0
6
2
6
— 1
1
0
.136
.6
.73
.32
.3
.26
.70
.16
.34
.50
.318
.07
.15
.61
.59
.10
.318
-------
Broun
Black
yellow
1A
2
31
74
95
111
154
200
4
9
19
22
38
51
62
71
80
91
112
113
114
118
122
3
5
N.R.
120 117
75.5 55
30.9 30
240 230
13.6
202 178
_
39.6 45
20.9
N.R.
303 853
2,400 3,050
22.7 31.4
_
-
304 370
-
_
_
-
_
-
Disperse Dyes
1,127 1,278
N.R. 34.1
-
-
-
_
7.51
-
-
8.52
-
-
-
-
-
_
2.08
1.43
-
1.08
1.34
1.00
3.06
14.0
0.85
0.125
8.85
16
-------
Orange
Red
7
23
44
50
1
3
5
13
17
18
20
25
30
38
1
5
13
17
44
46
54
65
72
73
1
2
- - 0
527 372 1
13
2
- - 0
61.4 50.9
100 22.7 2
12
120 59.1
1
14
210 224
- - 5
0
139 119
62.6 47.7
N.R.
97 70
- - 20
4
35
105 105
38
129
— — 9
67.3 N.R. 1
.34
.77
.4
.00
.20
.81
.5
.58
.0
.61
.60
.0
.85
.4
.5
.41
.92
17
-------
Black
152
Bed No.
Yellow No.
Orange No,
Bed No.
lellcu
Orange
Red
FD&C Dyes
2 474 441
4 N.R. N.R.
40 N.R. N.R.
5 561 504
6 464 369
D&C Dyes
4 3.18
6 4.08 N.R.
7 — —
9 16.4 N.I.
36 - 4.08
Mordant Dyes
1 7.72 N.R.
8 - N.R.
26
30
*3 MM —
22
r _ mm
17
-
-
_
-
-
-
-
-
-
-
_
-
6.49
0.114
2.95
1.00
0.91
1.02
Blue
6.56
18
-------
Gveen
Brown
Black
Yellow
Orange
Ned
29
47
1 11.8 18.2
21
33 22.3
40 - N.R.
11 248 200
17 45.4 68.6
79
Reactive Dyes
4
6
11
12
18
O _ _
5
7
4
6
7 -* -»
9 - -
12
13
15
0.10
1.00
0.114
1.59
-
-
16.1
0.095
2.73
0.20
0.50
1.00
5,51
0.20
0.25
1.43
2.65
0.97
1.85
2.00
0.90
11.0
7.36
4.40
-------
16 - - 4.31
17 - - 23.6
19 - - 3.50
21 - - 3.50
22 - - 2.90
23 0.97
24 - - 0.33
Violet 3 - - 7.70
5 - - 4.55
Blue 8 - - 41.9
10 34.2
13 - - 16.8
Broun 2 - - 8.85
Black 4 - - 6.56
Yellow
Solvent Dyes
2 10.0 N.R.
14 261 269
16
19
21
25
29
32
62
63
-
0.625
1.49
1.03
0.67
3.17
0.045
0.075
2.70
1.90
20
-------
65 - - 7.36
Orange 3 24.6 54.9 0.92
5 0.30
6 0.025
7 34.5 N.R.
9 - - 0.115
11 2.72
41 2.40
44 - - 0.45
45 0.025
Red 1 - - 0.10
3 - - 0.52
1 1.17
9 - - 0.50
12 0.015
16 - - 0.065
18 - - 7.56
19 0.50
24 - - 0.33
26 134 119
27 0.25
30 - - 4.84
36 0.065
90 - - 5.01
91 - - 4.93
92 - - 0.40
109 - - 7.95
-------
Violet
Blue
Brown
Blaok
Yellow
1
24
53
i — —
12 10.4 15
28
34
35
37
1 « —
? — —
3
6 - -
Pigments6'
1 784
3 180
12 2,550(4-1,486)
13 (See 17)
14 1,033(+1,385)
16
17 259 (Including
No. 13, others)
49
55
73
74 243
0.025
0.24
0.015
0.45
-
2.0
1.67
0.025
0.57
0.035
2.52
15.1
0.33
20,8
15.4
45.1
10.9
12.2
32.1
1.04
1.25
0.025
4.55
_
-------
Orange
Red
81
83
1
5
13
16
31
1
2
3
4
5
7
9
10
14
17
22
23
38
48
49 (Total)
51
52
53
54
—
-
-
182
65.4
153
-
85.8
28.2
738(+649)
140
38.6
_
-
-
-
30.4
60.8
88
60.4
1,230(41,090)
2,640(4-1,840)
-
790
1,100 (+85 5)
31.3
19.8
23.0
0.25
27.8
4.06
-
32.6
-
-
22.3
-
1.50
1.55
29.6
0.225
3.0
-
_
-
0.10
6.64
0.075
-
26.6
_
23
-------
57
63
68
112
119
144
144(<90I)
146
151
25
10
Blue
Green
Broun
Pigment Red 60)
)Lakes
Acid Red 26)
Permanent Red F4RH
(Toner)
465
19,1
87.6
69.5
126
15.7
5.09
2.0
32.5
0.045
25.0
135
8.50
4.50
7.26
2.5
a Listing is alphabetical by type of dye; within a type, order of color is
that used by Colour Index and U.S. Tariff Commission Reports. Units
are in metric tons.
b,c,d U.S. 1971, 1972, and imports, 1972, respectively; N.R. means Not
Released; a dash for a year preceeded or followed by a year repre-
sented by a number or N.R. means that the dye was listed in the pro-
duction figures section of the U.S. Tariff Commission Report, but
no figure was given. Except for Acid Violet 12 dashes for both
-------
years mean that the dye was not represented in the production
figures section.
e Left column is U.S. 1971, right one is 1972 imports. Figure in
parentheses is for the commercial forms.
There follows a copy of the listing of dyes whose production was
reported in the 1971 U.S. Tariff Commission Report; sulfur and vat dyes
were omitted from the end as these do not seem to contain azo linkages.
Confirmed azo dyes have been indicated by a dot centered to the right of
the dye's name. Production figures for those dyes marked with an asterisk
are given in Table 3. The next table (5) is a listing of the azo dyes
produced in 1971, but not in 1972. Then there is a table (6) of dyes
produced in 1972, but not in 1971. Finally there is a table (7) of
manufacturers, whose code names appeared beside the dye names. Tables
ii-7 are in the Appendix.
-------
In 1972 the U.S. Tariff Commission reported the following importation
of azo compounds under the designation of Benzenoid Chemicals and Products:
4-aminoazobenzene or Solvent Yellow 1 (4.5 metric tens); 4-aminoazobenzene-
disulfonic acid (23 metric tons); 4-aminoazobenzene-3,4'-disulfonic acid,
monosodium salt (13.6 metric tons); 2-aminoazobenzena-4',5-disulfonic acid
or Acid Yellow 9 (36.7 metric tons); azobenzene (58.6 metric tons); and
4,4'''-azobis(4-biphenylcarboxylic acid) or azo yellow acid (15.4 metric
tons).
The Chemical Week Buyers Guide 1974 Edition offprs for sale the follow-
ing azo compounds which are not to be found in the U.S. Tariff Commission
Reports under domestic production: 4-aminoazobenzene (Solvent Yellow 1)
and its hydrochloride; azobenzene; 4,4'-azobis(N,N'-ditiethylaniline);
azosulfamide (also known as Prontosil S or Neoprontosil) and which has the
chemical formula 2-(4-sulfonamidophenylazo)-3,6-disulfo-7-acetamido-
naphthol-1, disodium salt; 2,6-diamino-3-phenylazopyridine hydrochloride;
4-hydroxyazobenzene (Solvent Yellow 7); and Methyl Red (Acid Red 2).
In the Cyclic Intermediates section of the U.S. Tariff Commission
Reports are listed production figures for a few dyes, with manufacturers,
plus an alphabetical listing of dyes and dye intermediates apparently
lacking Colour Index names: Acid Yellow 9, 5.91 metric tons in 1971,
American Cyanamid Co., Nyanza Inc., Toms River Chemical Corp.; Food
Yellow 6, 141 and 207 metric tons in 1971 and 1972, respectively, Allied
Chemical Corp., A. Cyanamid, duPont, Toms River; Solveat Yellow 3, 148
and 180 metric tons, Alliance Chemical, Inc., A. Cyanamid, duPont, GAF,
Sterling Drug, Inc.
26
-------
Table 7a. Domestic Manufacture in 1971 and 1972 of Azo Intermediates
in the Dye Industry
8-acetamldo-l-(4-acetamido-2-hydroxy-5--nitrophenylazo) -2-naphthol, 1972,
Toms River;
3-[(2-acetamido-4-aminophenyl)azo]-l,5-naphthalenedisulfonic acid, 1972,
T oms Rive r;
5-amino-4,5'-dihydroxy-3,4'-[(2-methoxy-5-methy1-p-phenylene) bis(azo)]-
di-2,7-naphthalenedisulfonic acids 5'-benzenesulfonate, 1971, 1972, Toms
River;
2-(2-amino-5-hydroxy-7-sulfo-l-naphthylazo)-5-nitrobenzcic acid, 1971,
1972, Toms River;
m-[(4-amino-3-methoxyphenyl)azo]benzenesulfonic acid, 1971, 1972, duPont,
Toms River;
4-[(4-amino-5-methoxy-o-tolyl)azo]~4-hydroxy-2,7-naphthalenedisulfonic
acid benzenesulfonate, 1971, 1972, Toms River;
3-[(4-amino-5-methoxy-o-tolyl)azo]-l,5-naphthalenedisulfonic acid, 1971,
1972, Toms River;
7-[(4-amino-5-methoxy-o-tolyl)azo}-l,3-naphthalenedisulfonic acid, 1971,
1972, Toms River;
2-(4-amino-l-naphthylazo)-4-(1,1,3,3-tetramethyIbutyl)phenol, 1972, GAF;
m-[(p-aminophenyl)azo]benzenesulfonic acid, 1971 (duPont, Toms River), 1972,
Toms River;
7-[(4-aminophenyl)azo]-l,3-naphthalenedisulfonic acid, 1971, 1972, Toms River;
5-amino-8-(phenylazo)-2-naphthol, 1971, 1972, Alliance;
8-amino-5-(phenylazo)-2-naphthol, 1971, 1972, Alliance;
4-[(p-aminophenyl)azo]-l-naphthylamine, 1971, 1972, Allied;
5-[(p-aminophenyl)azo]salicylic acid, 1971 (Toms River), 1972 (Baychem
Corp., Toms River);
preceeding item, sodium salt, 1971, 1972, Allied;
m-(4-amirio-3-tolylazo)-benzenesulfonic acid, 1971, 1972, Totns River;
-------
3-[(4-amino-o-tolyl)azo]-l,5-naphthalenedisulfonlc acid, 1971, 1972, Toms
River;
7-[(4-amino-o-tolyl)-azo]-l,3-naphthalenediaulfonic acid, 1971, 1972, Toms
River;
3-(o-anisylazo)-benzensulfonic acid, sodium salt, 1971, 1972, Allied;
4',4'''-azobis(4-biphenylcarboxylic acid), 1971, 1972, duPont, Toms River;
3-(4-N-benzylamino-N-methylphenylazo)-l,2,4-triazole, 1972, Toms River;
4,4'-bis-[(p-hydroxyphenyl)azo]-2,2'-stilbenedisulfonic acid or Direct
Yellow 4, 1971, Toms River;
N-(2-chloroethyl)-4-(2-chloro-4-nitrophenylazo)-N-ethyl aniline, 1971,
1972, GAF;
N-[(5-chloro-2-methoxyphenyl)azo]sarcosine, 1971, 1972, Atlantic Chemical
Corp.;
N-[(5-chloro-o-tolyl)azo]sarcosine, 1971, 1972, Alliance, Atlantic;
dibenzylazodicarboxylate, 1972, Kay-Fries Chemicals, Inc., Wilson & Co., Inc.;
2-(5,8-dichloro-l-hydroxy-2-naphthylazo)-l-phenol-4-sulfonamide , 1972,
Toms River;
3-[(4'-N,N-diethylamino)phenylazo]-lH-l,2,4-triazole, 1971, 1972, Toms River;
'*,5-dihydroxy-3-(p-sulfophenylazo)-2,7-naphthalenedisulfonic acid,
trisodium salt, 1971, 1972, Eastman Kodak Co.;
\r,N!-[(3,3'-dimethoxy-4,4'-biphenylene)bis(azo)]bis(N-methyltaurine) , 1972,
GAF;
4-(a,a-dimethylbenzyl)-2-phenylazophenol, 1971, Toms River;
N,N-dimethyl-p-phenylazoaniline, 1972, Eastman;
l-(3,5-dinitro-2-hydroxy-phenylazo)-2-naphthol, 1971, 1972, Toms River;
2-[N-ethyl-p-[(6-methoxy-2-benzothiazolyl)azo]anilino]ethanol, 1971,
1972, Toms River;
N-[7-hydroxy-8-[2-hydroxy-j-(methylsulfamoylphenyl)azo]-l-napht.hyl]-
acetamide, 1971, 1972, Toms River
6'-hydroxy-5'-[2-hydroxy-5-nitrophenyl)azo]-m-acetotoluidide, 1971, 1972,
Toms River;
N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-l-naphthyl]acetamide, 1971,
1972, Toms River;
7-hydroxy-8-[(4'-[(p-hydroxyphenyl)azo]-3,3'-dimethyl-4-biphenyl)azo ] -
1,3-naphthalenedisulfonic acid, 1971, 1972, Toms River;
.1-(2-hydroxyl-l-naphthylazo)-6-nitro-2-naphthol-4-sulfonic acid, 1971, 1972,
Toms River;
l-(2-hydroxy-4-nitrophenylazo)-2-naphthol, 1971, 1972, Toms River;
°-[(p-hydroxyphenyl)-azo]benzoic acid, 1971, 1972, Eastman;
-------
3-[(4-(4-hydroxyphenylazo)-2,5-dlmethoxyphenylazo)]-benzenesulfonic acid,
1972, Toms River;
3-hydroxy-4-(phenylazo)-2-naphthoic acid, 1971, Inmont Corp.;
2-(o-nitrophenylazo)-p-cresol (OH = 1), 1972, Toms River;
p-phenylazoaniline (Solvent Yellow 1) and hydrochloride, 1971, 1972,
Allied, Am. Cyanamid, duPont;
4-(phenylazo)diphenylamine, 1971, 1972, Eastman;
4-(phenylazo)-l-naphthylamine, 1972, duPont;
5-(phenylazo)salicyclic acid, 1972, Toms River;
N-(p-tolylazo)-sarcosine, 1971, 1972, Blackman-Uhler Chemical Co., GAF;
4~(o-tolylazo)-o-toluidine hydrochloride (Solvent Yellow 3 hydrochloride),
1971, 1972, GAF;
4-(2,4-xylylazo)-o-toluidine> 1971, 1972, Allied;
4-(2,5-xylylazo)-o-toluidine, 1971, 1972, Am. Cyanamid;
4-(2,4-xylylazo)-2,5-xylidine, 1971, 1972, Allied;
4-(xylylazo)xylidines, mixed, 1971, 1972, GAF.
-------
Finally the U.S. Tariff Commission reports that duPont is the only
producer of azobisisobutyronitrile (giving no production figures) and
that azodicarbonamide is produced by Fairmount Chemical Co., Inc., Stepan
Chemical Co., and Uniroyal, Inc. (again giving no production figures for
1971 or 1972, but indicating that 2,060 metric tons were sold in 1971).
III. USE
Azobisisobutyronitrile
Nowak and Rubeus in Kirk & Othmer (1969) stated that this compound -
NCC(CH3)2N = NC(CH3)2CN - was the best known azo compound for free
radical initiation of the polymerization of polyesters to resins, but
had not yet come into wide commercial use.
Ito (1969) reviewed the properties pertinent to use as a foaming
agent for plastics.
La Clair in Modern Plastics Encyclopedia 1972-1973 commented that
AIBN, as it is commonly abbreviated, had some use as s chemical blowing
agent for polyvinyl chloride foamed plastics, and tha : it required pre-
caution in handling.
Azodicarbonamide
The Federal Register (1962) announced that this chemical,
H2NC(0)N = NC(0)NH2, had been approved as an aging and bleaching agent
in white and whole wheat flours. The limit for such use has since been
set at 45 ppm.
Ito (1969) reviewed the properties pertinent to use as a foaming
agent for plastics.
-------
La Glair in Modern Plastics Encyclopedia 1972-1973 commented that
it was used as a chemical blowing agent for HDPE, PP, PS, and PVC
foamed plastics. It was considered to be non-toxic, self extinguishing,
and of excellent storage stability.
Azo Dyes
As may be seen from the preceeding section on Production, the usage
of azo dyes has been increasing steadily. For many of the major end
uses such as natural and synthetic fabrics, paints, plastics, and print-
ing inks, use of any particular dye or color can be highly dependent
upon the dictates of fashion, price competition in substrates, packaging
material changes, etc. Use in photographic film and enlarging paper is,
presumably, more insulated from such "extraneous" concerns. No published
breakdown of end use of azo dyes was found.
Usage of azo dyes for foods, drugs, and cosmetics is subject to
increasingly intensive toxicological studies; these have resulted in
curtailed or discontinued usage for many dyes. These FD&C dyes in a
lower purity grade nay have uses in general manufacture of dyed goods.
There is no agreement between the United States £>nd Europe in the
dyes allowed into and upon the human body. The U.S. is much more restric-
tive. Bigwood (1973) has analyzed the reports through 1972 of the Joint
FAO-WHO Expert Committee on Food Additives. This committee had examined
244 natural and artificial additives with the intent of determining an
"acceptable daily intake" in units of mg/kg of body weight. Bigwood came
to the conclusion that there had been some hedging in basic definitions,
and also raised the important point that it is extremely difficult to
determine how much of a particular additive is being ingested except on
an individual basis. Only six azo dyes had been studied. A temporary
31
-------
upper limit of 0.75 mg/kg of body weight had been set for amaranth and
Ponceau 4R; Citrus Red No. 2 was not to be used as a food additive;
no decision had been reached on Black 7984, Brilliant Black BN, and
Orange 1.
Collins and McLaughlin (1972) indicated that about 680 metric tons
of amaranth was in annual use in foods, drugs, and cosmetics in over 60
countries.
The newest dye on the FD&C scene seems to be FD&C Red No. 40, which
was announced in the 1971 Federal Register. The specifications for food
usage once again emphasized the fact that dyes are mixtures and are not
discriminated against as such.
Carriere and Luft (1966) examined then current lists of regulated
dyes in the U.S.A., W. Germany, France, and Italy, and combined them in
tabular form for comparison of all the dyes mentioned in at least one of
the country's lists - the Fr&nch and Italian being non-authorized. These
are reproduced here as Table 8.
-------
Table 8. Comparison of U.S. and European FD&C Dye Lists
REDS
Colour
Index Xo,
16,185
45.430
14.700
16.150
15.850
15.850
15.500*
26,100
26.125*
45.380
45.380
45.366
45.410
45.410
45.457*
73.360
15.800
15880
12.120*
12085
12.350*
13.058
18055*
16.105*
15.620
14 720*
18.050*
27.290
12.141*
16.155
15.585
15.585
15.630
15.630
15.630
15.630
45,170
17.200
45.170
14.780
14.830
15.580
16.250 '
18.000
18.020
18.025
45.360
45,380 !
45.38S j
45.400 ;
45,405
45.425
45.435 '
45.440
58.000
16.180
27.306 i
45.IGO
58.005 1
14.895 '
27.300
68.000
16.050
12.315 ;
45.510
16.255
16.290
, — i
18.810
14.830
,
16.0 JS
26.105
18.0G5
50 240
j Federal Designation
FD & C Red No. 2
3
; Ext. V&C Red No. 24
D & C Red No. S
6
7
14
17
18
21
22
24
27
Use in U.S.A.
i A
1 A
A
B
B
B
B
! B
B
B
B
B
B
28 B
29 I B
30 I B
31 i B
34 ! B
35 i B
36
B
38 B
39
B
Ext. B & C Red No. 1 : C
2
8
C
C
10 : C
11 | C
Usu in Germany
I^Rot 3
C-Rot 38
C-Kot 5
C-ext. Rot 35
C-Rot 12
C-Rot 12
—
^
C-Rot 30
C-Uot 30
—
C-Rot 34
C-Rot 34
~"
C-WR Rot 12
—
C-Rot 14
C-ext, Rot 1
French No.
Al 500 E
A1551E
519
515
542
542
535
544
533
547
- — •
549
548
548
514
524
530
503
532'
C-Rot 1 ! 520
C-ext. Rot 4 105
C-Rot 4
C- wet. Rot 22
C-ext. Rot 19
526
527
509
C-ext. Rot 18 | 523
— 1 Al 502 K
C-ext. Rot 21 1 528
13 1 C ; C-cxt. Rot 24 | 507
14
C —
15 ! C
D & C Red No. 8
Cx, D, E
9 ! Cx. D
10 i Cx, D
11
12
Cx, D
Cx, D, E
539
— ! 516
Use in
France
0-1-2
0-1
0-1-2
1-2
1
1
1
1-2-3
2-3
1
—
1
1
1
1
1-3
1
1
1
1
1-2
1-2
2
2
2
Use in
Italy
-f
+
+
+
+
+
+
+
+
-f
+
+
+
+
+
2 I +
2
2
+
4-
2-3 i
0-1-2 i
C-ext. Rot 17 j 534 i 1 j +
C-ext. Rot 17 ; 534 1 1 j + ;
C-ext. Rot 33 1 536 1 1
C-cxt. Rot 33 i 536
C-ext. Hot 33 ! 536
13 1 Cx, D C-ext. Hot 33 i 536
19 : CX, I). E
C-cxt. Rot 27 ! 521
33 '. Cx, D, E C-\VK Hot 2
37 | Cx, E
— I . —
. —
—
_
—
—
—
—
—
—
—
—
—
. —
—
—
—
—
—
—
—
—
—
C-ext. Rot 27
C-Rot 6
C-Rot 7
C-Rot 9
C-Rot 19
512
545
C-Rot 21 !
C-Rot 22 i 501
1
1
1
1-2-3
1-2
1
+ :
+
+
+
1 1
— i C-Rot 23 ' !
— -
—
—
—
—
—
—
—
—
—
—
—
i __ I
" ° !
-
~. . ',
— —
—
—
— .
—
—
..
,
—
—
—
_
. —
C-Rot 25
C-Rot 30 .
C-Rot 31 |
C-Rot 32
C-Rot 33
C-Rot 3S
C-Rot 36
C-Rot 37
C-Rot 4 1
C cxt. Hot 20
C-ext. Rot 25
C-ext. Rot 26 !
C-cxt. Rot 31 1
C-NVR Rot 1
546
550
j
506
505
C-WR Rot 5
C-\VR Rot 10 1
_
-
I^Rot4
1. Rot 5
—
1
508 i
511 1
513
A1517E
518 E
522
525
2
2
2
2
|
2
1-2
2
+
+
+
0-1 2 +
1-2 [ +
2
2
— — 529 2
- i — , 531
1 - ! 537
"
I
s;w
540
I • Ml
2
2 : f
23 \
i :
?.
j ,'tt'l [ 7.
MO
'i. i
-------
ORANGES AND YELLOWS
Colour
Index No.
45.425
45.425
45.456'
45.371*
11.725*
14. GOO
12.100'
I'edci.il Designation
I) & C Orange No. 10
11
14
16
Kxt. D & C Orange No. 1
3
4
15.510 i D & C OrariKe No. 4
45.370
12.075
19.140
15.985
47.005
47.000
13.065
18.820
1 1 .680*
14.010*
10 3!6
45 350
45 350
45.365
58,000
16 230
60.515
77.199
1 1 .920
15.575
15.970
45.395
71.105
11.710
11,730
12.775
12.780
13 900
18.950
48 055
49 005
18.736
18745
18 690
. —
—
48.035
48040
29.020
29.025
65.405
65.410
68.420
40215
59.700
5
17
V. 1> iV C Yellow No. S
6
10
11
Kxt 1) \ C Yellow No 1
3
5
6
7
11
12
. — .
—
—
—
—
—
—
_-
. —
—
—
—
—
—
—
—
. —
—
_
—
—
—
—
. —
—
—
—
—
_
—
—
—
69 025
69 540 i' —
' 13.015
14.270
75 300
41.000
25.135 !
11.380 1 —
1 1 .390 I —
10.315
19.130 j
1
43 35)5 j
16.02(1
Use in U.S.A.
B
B
B
B
C
C
C
D-E
D-E
D
A
A
R
B
C
C
C
C
C
Cx
Cx
—
—
—
. —
, —
,
. —
—
, — :
,
— -
— i
„
. —
. — .
. .
.
—
—
. —
f
—
~
—
. —
, —
. —
«~..
—
—
—
—
I 'so in
C-Kot
C-Ko
C. pxt
C Ora
C-ext
L-Gcl
L- Or;
L-Gel
C Gel
C-ext
C-c-xt
C-ext
C-ext
C-cxt
C-ext
C-Kot
C-Kot
C-Ont
C- Gel
C-Gel
C-Ora
C-Ora
C-Ora
C-Ora
C-Ora
C-cxt.
C-ext.
C-ext.
C-cxt.
C cxt.
C-ext.
C-ext.
C-cxt
C-cxt.
C-ext
C-ext.
C-cxt.
C-ext.
C-ext.
C-ext.
C-WI?
C-WIi
C-Wli
C-AVR
C-WR
C-\VR
C-WI?
C-Wli
C-AVK
I.-tk-l
L (".el
t f'f,\
l.-LiCl
1,-Gell
_
-
-
Geraiany
t 37
t37
. —
. Gelb 4
'
inge 2
. Hot 34
Ib 2
»ngc 2
Ib3
Ib4
. Gclb 10
. Gelb 12
. Gclb 2
. Gclb I
. Gelb 16
. Gclb 16
t 26
t 41
tnge 5
b6
1)7
ingc 1
mge 3
uigc 4
tngo 7
ini!c 8
. Gelb 3
. Gelb S
. Gelb S
. Gclb 9
. Gclb 11
. Gclb 13
. Gclb 18
. Gclb 19
. Orange 1
. Orange 2
. Orange 3
. Orange 4
. Orange 5
.Orange 6
. Oranqi; 7
I Gclb 1
< Gelb 2
? Gelb 3
{ Gclb 4
< Gclb 6
i Orange 1
? Orantjo 2
6
1>7 ]
—
—
—
_
—
French No.
404
406
413
401
410
407
412
408
402
411
At 21 7 E
At 203 E
208 E
A1215E
211
209
205
212
202
202
403
400
409
213
218
Al 201 E
200
204
206
207
210
214
216
405
4,4 i
Use in
France
1-2
1-2
1
1
2-3
2
2
1-2
1
1-2-3
0-1-2-3 ]
0-1-2
2 j
1-2
2
2 i
2-3
-2-3
-2-3
-2-3
2
-2
-2
2
2
1-2
1-2
2
2-3
2-3
2
2
2
1-2
2
Use 1:1
luh-
-------
GREENS AND BLUES
Colour
Index N*o.
42.085
42.095
42.053
61.570
61.565
42.100'
59.040
10.020
42.090
42.090
73.015
73000
42.052
69.825
52.015*
63.010'
42.045
42.735
42.750
42.755
42.135
43.820
44 0-10
44.045
44.075
60.730
62.085
63.000
74.180
77.007
77.510
42 170
62 550
42.080
42.140
50.315
50.320
52015
63.010
64.505
74,160
(74.180)
10.006
42.040
42.050
74.260
34.140
34.230
62.105
70.305
34.270
77.288
69.800
42.051
75810
75.810
61.530
—
42.770
43.535
61.555
69.SIO
51.175
42 052
61.525
73.000
50.405
42.000
52.020
44.025
Federal Designation
F, D & C Green No. 1
2
«
D & C Green Xo. S
6
7
8
lixt. U&C Green No 1
V. D A C Bhu- No 1
L> & C IHuc N.i -I
F, D * C Ulis, No. 2
D & C Blue No. 6
7
9
Ext. D & C Blue No. 1
4
__-
—
.
—
—
—
_.
_ -
_—
—
—
—
—
—
. —
—
_
,
. — -
„.
—
—
— .
—
—
—
—
__
—
*
—
. —
—
—
—
~- .
_ .
—
—
--
—
^-
—
—
, —
— .
—
._
—
_
Use in U.S.A.
A
A
A
B
B
B
B
C
A
B
A
B
B
B
C
C
Use in German)
,
—
C-Grim 5
C-ext. Grun 4
C-Grun 2
C-Gc-lb S
C-Grun 1
—
—
L-Hlau 2
—
—
C~ext, Blau 6
C-ext, Blau 8
C- Blau 2
C-Klau 3
C-Bl-AU 4
C-Blau 5
C-Blau 6
C-Blau 7
C-Blau 8
C-Blau 9
C-Blau 10
C-BIau 11
C-Blau 12
C-Blau 14
C-Blau 15
C-Blau 16
C-Blau 17
C-Gnin 3
C-Grun 6
C-ext. Blau 1
C-cxt. Blau 2
C-ext, Blau 3
C-ext. Blau 4
C-ext. Blau 6
C-ext. Blau 8
C-cxt. Blau 9
C-cxt. Btau 10
C-cxt. Blau 11
C-cxt. Grim 1
C-ext. Grim 2
C-ext. Griin 3
C-ext. Griin 5
C-\VR Blau J
C-WR Blau 2
C-VVU Blau 3
C-WR Blau 4
C--WK Griin 1
C- \VKGriin2
L-Blau 1
L-BIau 3
L-Grtin 1
L- Grun 2
French No.
711
712
710
701
709
703
700
708
7
7
A119E
2
3
14
17
12
8
702
704
1
4
5
6
9
10
11
13
15
18
20
705
706
707
Use in
France
0-1-2
0-1-2
0-1-2
1-2-3
1-2-3
1-2
1-2
o
1-2-3
1-23
0-2
2
2-3
2-3-4
2-3
1-2
2
1-2
2
V
0-2
2
2
2
2
1-2-3
2
1-2
2-3
1-2
2
2
2
2
Use in
Italy
+
+
+
+
+
+
+
+
+
+
+
+
+
i
-[-
!_
-r
T
-r
•f
+
-------
VIOLETS, BROWNS, BLACKS
Colour
Index No
42.G1D
60.725
60 ?:<(>
20.170
20,4"y 0
45.19,)
45 SCO
42 571
165KO
43,525
61.7)0
42 555
42 (Bii
4?. b.; ')
42 M.i
4274;>
21.010
2!. 010
20 :t(|0
21 ono
12.4rf()
13 OS.i
20. tVd
25.ii to
27. 24 S
35.S70
14.HU5
28.4 W
Federal Designation
F, D & C Violet No. 1
D & C Violet No. 2
Ext J)<\:C Violet No. 2
1) & C i;r,»wii Mo. 1
1) ft C Black No. 1
—
—
—
—
—
~ —
—
—
—
—
„_
..-.
.. _
__
—
,
_.
,. -
,
: '
Use in U.S.A.
A
B
C
B
B
—
—
—
,
~—
.
~
. — .
—
—
, —
—
—
—
—
—
—
— ,
, —
„
_
_—
,
• —
Use in Germany
C-Violett 4
C-Violett 7
C-BIau 11
French No.
804
801
800
O-Kraun 2 I 104
OWR Schwarz 1
C-Violctt 6
C-cxt. Rot 26
C-Violett 3
C-Violctl 1
C- Violctt S
C-Violett 8
O-Violctt S
. —
C-cxt. Violctt 6
—
—
C-cxt. Violctt 5
—
C-cxt. Violctt 7
, —
—
_—
. —
C-cxt. liraun 1
C-cxt. Itnuin 2
C-AVU Schwarz 1
C~\VKSehw;ir/2
C-WU Schwarz 3
C-\VK Schwiirz 4
C-WH Hnuiit 1
L- Sdiwnr/ 1
302
803
802
805
—
—
—
—
806
808
809
810
811
812
—
100
101
102
lot*
. —
—
„
, —
—
« —
„
Use in
France
0-1-2
1-2-3
2
1-2
1-2
2
1-2 .
2
—
, —
. —
— -
2
2
2
*>
2-4
2
__
2-4
2-4
4
4
, —
—
—
. —
. —
, —
-~~
, —
I'SO ill
Italy
,N«te$ omf Abbreviations
(I) Use in U.S.A.
A : F, D & C colours, unrestricted use
B ; D & C colours, unrc.sliict<:d u$e
C ; external 1> sV C colouis
C% ; I) & C colours, restricted usage, but unrestricted for external D & C usage
D : D & C colours restricted to 6 per cent maximum (pure dye basis) for lipstick Use
E : D & C colours restricted to 0-75 ing. maximum mgestion per day, tor preparations such as roouthwashes and dentifrices
• Recently dciistetl by the F.D.A. (<:/. H. D. Gouldcn in "Drug and Cosmetic Industry" of February 1965).
(2) Use in Germany
I. . food colour, unrrstricti.-d use in cosmetics •
C : unrestricted use in cosmetics
C-cxt. : for external use only : not necessarily safe for ingcstion
C— "WK : for use m uaslunp and nn.sing, or as a solvent or propcllant, provided that the material has only transient appiicati
not necessarily sal« wlien ingested or remaining on the skin.
(3) Use in France
0 ; completely acceptable for use in any cosmetic including those likely to be ingested {e.g. dentifrices or in mouthwaslies)
1 : for cxteinal use (lijHlieUs included)
2 : extcnial colours in the F.I),A. sense of the wotd
3 ; for soaps and non-soapy detergents »
4 : for hair preparations
Al : mentioned in "alimentaire decret du 25.3 58"
E : mentioned in "C.E.E. alimentaire d&ret du 11.11.62"
(4) Use in Italy
+ : mentioned by F. GhiiOtti
Reprinted with
Perfum. . &
permission from Soap,
39:29-34 (196r!fr
Copyright by United Trade Press Ltd
Food Technology (1968) presented a report by Hazelton Laboratories
on the use of FD&C colors in food. Tables 9 and 10 here are taken from
36
-------
this report and indicate the major food categories which use colorants,
and amounts of colorants used in them, respectively.
Table 9- Major categories of processed food which use certi-
fied FD&C colors in their manufacture, and color concentration
levels employed.
Color Concentration, ppm
Category
Candy and Confections
Beverages (Liquid & Powdered)
Dessert Powders
Cereals
Maraschino Cherries
Pet Foods
Bakery Goods
Ice Cream and Sherbets
Sausage (Surface)
Snack Foods
Meat Stamping Inks
Miscellaneous
Range
10-400
5-200
5-600
200-500
100-400
100-400
10-500
10-200
40-250
25-500
5-JOO
Avenge
100
75
140
350
200
200
50
30
125
200
Tables 9 & 10 reprinted from Food
Technology/Journal of Food Science,
Vol. 22, 1068. Copyright 0 by
by Institute of Food Technologists.
Table 10. Pounds of primary colors used in foods, drugs and cosmetics. Figures represent sales for the first nine months of 1967
and do not include exports or sales to jobbers and other manufacturers.
Candy, Confection
Beverages
Dessert Powders
Cereals
Maraschino Cherries
Pet Food
Bakery Goods
Ice Cream, Sherbet,
Dairy Produce
Sausage
Snack Foods
Meat Inks
Miscellaneous
Subtotal
Pharmaceutical
Cosmetics
YELLOW
No. 5
•
59,903
78,933
59,961
52,496
5,644
101,743
77,885
35,048
6,502
18,456
15
44,841
541,427
17,275
3,125
YELLOW
No.6
•
52,770
181,292
51,622
35,464
4,830
23,226
42,203
23,868
99,605
11,409
0
29,134
555,423
15,938
2,148
RED
No. 2
•
67,637
282,695
62,363
15,558
8,104
67,058
43,522
29,697
36,084
3,623
12
46,219
662,572
21,179
3,417
RED
No. 3
1 1 ,665
1,056
8,616
1,421
3,469
1,023
9,560
621
4,970
766
10
18,200
61,377
12,168
903
RED
No. 4
•
0
0
0
0
1 1 ,308
0
0
0
0
0
0
398
1 1 ,706
1,186
630
BLUE
No. I
6,632
15,800
3,270
843
597
1,473
3,680
2,599
647
305
11
5,345
41,202
3,250
397
BLUE
No. 2
2,499
2,375
1,659
99
0
6,764
673
179
0
0
0
1,990
16,238
593
30
VIOLET
No. 1
1,459
985
0
0
0
1,278
369
45
0
2
2,223
1,134
7,495
347
96
GREEN
No. 3
124
301
14
0
98
0
7
7
0
0
0
1,298
1,849
220
27
ORANGE
B
•
0
0
0
0
0
0
0
0
16,890
0
0
0
16,890
0
9
TOTALS
202,689
563,437
1 87,505
105,881
34,050
202,565
1 77,899
92,064
164,698
34,561
2,271
148,559
1,916,179
72,156
10,773
TOTALS
561,827 573,509 687,168 74,448 13,522 44,849 16,861 7,938 3,096 16,890 1,999,108
Note: To convert to metric tons, move decimal three places to the left
and multiply by 0.453.
Azo dyes are indicated by a • below the name
The Hazelton report commented that the azo dyes Yellow 5 and 6 and Red 2
comprised about 90% of the total of all food dyes.
Vodoz (1970) in an article on European food additives presented a
-------
Table II—Food colors permitted (+) in European countries.1
COLOR
REDS
Ponceau MX
Ponceau 4 R
Carrnoisfne
Amaranth
Red 10 B
Erythroiine BS
RedZG
Red6B
RedFB
Ponceau 3 R
Fast Red E
Ponceau 6 R
Scarlet GN
Ponceau SX
Acid Fuchsine S
ORANGES and
YELLOWS
Orange G
Orange RN
Orange GGN
Oil Yellow GG
Tartraiine
Naphthol Yellow S
Yellow 2 G
Sunset Yellow FCF
Oil Yellow XP
Acid Yellow
Quinoline Yellow
Chrysoin S
GREENS
Green 5
Guinea Green B
Fast Green FCF
BLUES
Blue VRS
Indigo Carmine
Indanthrene Bl. RS
Patent Blue V
Brilliant BL FCF
VIOLETS
Violet INP
VioIer5BN
Violet 6 B
BROWNS
Brown FK
Chocolate Br. FB
Chocolate Br. HT
BLACKS
Black PN
Black 7984
C.I.
No.
* 16150
• 16255
• 14720
• 16185
• 17200
45430
•18050
t 18055
• 14780
• 16155
• 16045
• 16290
•14815
• 14700
• 16230
• 15970
• 15980
• 11920
* 19140
10316
* 18965
"15985
• 12740
•13015
47005
• 14270
44090
42085
42053
42045
73015
69800
42051
42090
42580
42650
42640
41 mnuaaa—
_
• 20285
• 28440
» 35445
Toxleot. evaluBtion
WHO techn. rep.
ier., 309, 1965-"
C,
c,
c,
A
C3
B
C.,
D
D
E
c»
C,
c,
E
C,
C,
D
c,
—
A
c,
D
A
D
C,
C,
c,
c,
E
B
c,
B
C,
c",
B
_
c,
c,
c,
D
C,
c,
c.
COUNTRIES
- 1 1
Illlfl^l
+ +*
-|_ -|_ 4, 4, 4. 4- 4-
4. 4- 4. 4. -|- -f 4.
4-4.4.4.4.4.4,4.
+ +
4- -f. _(. 4. 4. 4- 4.
+ +
+ + +
+ +
4.
4.-)- 4. 4. 4. 4, 4.
4. 4. 4. 4. 4.
4-4.4- 4.4. 4.
4. 4.
+
+ +
+ - +
4. 4. 4. 4. 4-
4.
4.4^4.4.4.4.4.4.
+
+ +
4. 4. 4. 4. 4. 4- 4.
+ +
+ + + + + +
+ + + + +
+ +
+ +
+ +
4.4. 4.
+ +
4.4.4.4.4.4.4.4.
4- 4. 4. 4. 4.
+ + + +
+
+ +
4,
+ + +
+ +
4- +
+ +
+ + + + + 4 +
+ +
EEC numbers
124
122
123
127
126
125
111
102
no
105
104
103
142
132
130
131
151 '
152
.f.E.C.
COUNTRIES
I 51
i * 2 £ H
I ! * $ I 1
« S * £ s *f
BQ tfa ffi — — * ~
+ + + + + +
++++++
+ + -r + + +
4. 4. 4. 4- 4. 4.
4. 4. 4. 4. 4- 4.
4 4. 4. 4. 4. 4.
+ + + + + 4
+ + + + + +
4. 4. 4 4. 4. 4.
+ + + + + +
4. 4. 4. 4. 4. 4.
+ + + + + +
4. 4. + 4.
+ + + + + +
4. 4- 4. 4. 4. 4.
4. 4. 4. 4. 4. 4.
+ + + 4 + +
r *>
.£ 3£
o. 5
4- 4-
4-
+ +
-(- 4-
4.
4-
4-
4-
+
4. 4.
+ 4
4-
4.
+ +
4.
+
EASTE !K
COU^Tfllkl
5 S
£• » *• .1 ~
; s I c 'S '-
E s, f 5 e -J
O to X eC i£ u
+ + 4 -r
4. + -1
+ + + 1 -> i-
4-4- 4-
+ 4
+ +
4. 4.
+ +
+
4. 4. 4. .(. 4. -|
+ -1-4-
4. 4, 4.
I
1
+
4- 4. 4 4. 4. 4
+
4- 4-4. 4.
1 Se» 8.F.M.I.R.A. Information SHeet 261, 1969; «nd "food Creee»in9 §nd Paclnging Directory I96f-70 "
** = *<"P»«W« •« » *n »fc»i.»«bi intuffir ln<
fic»tion C, =r Th« d<)> ire intwffaient lor • 'mil «*i)ujrion; how«v«r, long. term to«idfy l.il. h*y* l>. ti> cJon« C, = long l"r
1iS« iiit> >r> not luffidtnt for in •»«lu»tlon, but th»f» i«mi to bt • ritk of nouvity, 0 No loxicolovi'ol d«t» "I «ll. I Tr.
lhu»d
l (o
m'eftr
l(.r it
-------
table of dyes used in more countries than was given in Table 8 above;
his table is reproduced here as Table 11, with the azo dyes denoted by
a • to the left of the Colour Index number.
Noonan (1972) reported in Handbook of Food Additives that the then
current list of FD&C azo dyes consisted only of Red Nos. 2, 4, and 40,
Yellow Nos. 5 and 6, and Orange B. Of these Red No. 4 was restricted
for use in maraschino cherries, and Orange B restricted for sausage casings,
Miscellaneous
Shibata (1972) has written an extensive review of the use in the
analytical chemistry of polyvalent metal ions of the family of azo com-
pounds derived from the parent l-(2-pyridylazo)-2~naphthol(PAN).
IV. CURRENT PRACTICE
No information was discovered.
V. ENVIRONMENTAL CONTAMINATION
Bartha and Pramer (1967) were the first to report finding 3,3',4,4'-
tetrachloroazobenzene in soil which had been treated with the herbicide
3,4-dichloro-N-propionylaniline (propanil). Since noi.e of the azo com-
pound could be detected in sterilized soil treated with a sterilized
solution of the herbicide, the authors concluded that soil microorganisms
were at least partially responsible for the transformation. The authors
could not find any published reports on the biological activity of the
azo compound.
Bartha et al (1968) concluded that peroxidase was the catalyst
responsible for the transformation of various chloranilines to chloroazo-
benzenes in soil. They compared the products resulting from treating
chloroanilines in vivo in soil and in vitro in buffered H202 containing
-------
orseradish peroxidase type II. The results are In Table 12.
Table 12. Formation of Azo Compounds from Anilines
in Soil and by Peroxidase
CHLORO-
SUBSTITUTION
0
2-
3-
4-
2,3-
2,4-
2.5-
2,6-
3,4-
3, 5-
2,4,5-
2,4,6-
TRANSFORMATION RMIM''E TRANSFORMATION
IN SOH- HUILiyE BY PEROXIOASt
a
^N»N^) _ .Q*H,-~ G^N^N-^
"a a ^i ' a
ci ci a
ci-Q-M-N-O^ — c,-O*H,— C,-^N-N^>C,
^N.ff.^1 ._ q^Ht_ ^
CI ci CI CI
0-Q-*-N-£}-CI *_CI^Q-NH,— 0
CI ci CI
none •*— O"NH«~ - none
none — - ~CI
ct' ci ci^
QHN-N-Q> — Q^NH,— •» none
a ci «'
none •* — CI"G^Hi — •- none
none — CI-Q-NH.— ~ none
a
Reprinted with permis-
sion from Bartha e_t al. ,
Science 161:582-81"
(1968). Copyright bv
American Associatioi toi
the Advancement of
Science.
a - Unidentified non-azo aromatic reaction product
b - No reaction occurred
Belasco and Pease (1969) were not able to detect 3,3',4,4'-
tetrachloroazobenzene in soil which had been treated for 12 consecutive
years with the compound l,l-dimethyl-3-(3,4-dlchlorophenyl)urea (diuron)
at 224.6 or 449.2 mg/sq.m. They were able to confirm Bartha and Pramer's
initial finding with propanil, at application rates of 250 and 500 ppm
(the latter equivalent to 56.15 g/sq.m to a depth of 1.7. cm), in the
-------
laboratory. However, diuron at 500 ppm gave no detectable azo compound.
They did not believe that any azo compound formed derived primarily from
an aniline precursor.
Bartha (1969) found that soil treated with the herbicides propanil
and solan[N-(3-chloro-4-methylphenyl)-2-methylpentanamide] could produce
an azo compound derived from portions of both, namely 3,3',4-trichloro-
4'-methylazobenzene. This azo compound also resulted from treating soil
with 3,4-dichloroaniline and 3-chloro-4-methylaniline. Bartha indicated
that because of unequal degradation rates of the two herbicides, the
mixed azo compound was unlikely to be produced if propanil treatment pre-
ceeded that by solan.
Kearney et al (1970) examined ground used to grow rice and treated
with propanil at various recorded times. They found 3,3',4,4'-tetra-
chloroazobenzene at a level of <0.2 ppm in the top 10 cu of soil treated
at the rate of 6.7 kg/hectare.
Sprott and Corke (1971) tested the formation of 3,3',4,4'-tetra-
chloroazobenzene from 3,4-dichloroaniline in two loamy and two clayey
soils under varying conditions of water and oxygen cor tent of the soil.
The soil which produced the most azo did so at an optimal temperature of
25°C, an optimal aniline concentration of 500 Vg/g, and aerobic atmosphere.
In general very little of the aniline which disappeared was converted to
the azobenzene, at best only 4.8%. Degradation of the azobenzene in the
soils was relatively rapid.
Bordeleau and Bartha (1971) tested the ability of two common soil
fungi Penicillium piscarium and Geotrichum candidum to produce tetra-
chloroazobenzene from propanil. P. piscarium by itself could not metabo-
lize propanil past the dichloroaniline stage. G. candidum by itself
could not metabolize propanl] at all, but rould rrjnvert the anJIinf I o
-------
the azobenzene. Together they were able to produce the azobenzene from
propanil. The only benefit to the fungi seems to be a reduction in the
toxicity of their environment, the azobenzene being less toxic than the
propanil or the aniline.
Rosen and Siewierski (1971) prepared a derivative of 3,3',4,4'-
tetrachloroazobenzene in which one of the P-chloros has been replaced by
the nitrogen of 3,4-dichloroaniline. This derivative had earlier been
proposed as a degradative product of propanil. The authors demonstrated
that the derivative was stable after at least two months in soil capable
of converting the aniline to the tetrachloroazobenzene; the derivative
in methanol was resistant to two weeks of exposure tc glass-filtered
sunlight, and to ten hours of exposure to UV light of wavelength over
297 nm.
Helling (1971) found that the Rf values in a soil sample were 0.24
for propanil, 0.22 for 3,4-dichloroaniline, and 0.00 for 3,3',,4,4'-
tetrachloroazobenzene.
Briggs and Ogilvie (1971) reported that 3-chloro-4-methoxyaniline
was converted in soil into 3,3'-dichloro-4,4'-dimethoxyazoben2:ene by a
free radical mechanism.
Child et al (1972) reported that 3,3',4,4'-tetrachloroazobenzene
was optimally effective at the 81 mg/kg level against aaenocarcinoma
tumors in mice, nearly tripling the survival time of animals receiving
mammary tumor transplants.
Burge and Gross (1972) found that 3,3'-dichloro- and 3,3',4,4'-
tetrachloroazobenzene were satisfactorily extracted from a variety of
soil samples with 95% ethanol. Analysis by gas-liquid chromatography
using a micro-coulometric detector did not require evaporation of the
alcoholic extract.
1.2
-------
Kearney and Pliimner (1972) studied the effect of 3,4-dichloroaniline
concentration in soil on tetrachloroazobenzene generation. Between 1 and
100 ppm there was about a twofold increase in azobenzene for each tenfold
increase in aniline; between 100 and 1000 ppm there was only a 10% increase
in azobenzene. They also seemed to have isolated both cis and trans forms
of the azobenzene by thin layer chromatography.
Bordeleau et al (1972) studied the conversion of 3,4-dichloroaniline
to 3,3',4,4'-tetrachloroazobenzene in a H202~peroxidase system with the
intent of determining the intermediates. Their results are represented
in Figure 1. The main pathway involved intermediate (II), but there was
evidence for the presence of the free radical (III).
J,4- OICHlOROAMUNE (I]
01 Nrt
I
•OH
I
4 A"
\B<
\
H
• > HO-N
/
B EXCESS (I]
(Ct)
«o-v/°
CZ)
Tigurc I. Proposed pathway of 3,3',4,4'-(c(rachloroa7.obc
-------
VI. MONITORING AND ANALYSIS
A. Analysis
Spain and Clayton (1955) analyzed for dyes in feces, tissue, and
urine by alkaline digestion, acidification, filtration, colorimetric
measurement, reduction with stannous chloride, and remeasurement of
color.
Parsons and Seaman (1955) described a method of external genera-
tion at a mercury pool electrode of the Ti(III) used to titrate azo
dyes. Comparison of this coulometric titration with the standard
titration with Ti(III) using technical grades of Orange II, tartra-
zine, and p-aminoazobenzene was favorable..
Feigl and Neto (1956) described a spot test for azo dyes whose
structures incorporate a p-phenylenediamine or a p-nitroaniline
moiety. Fusion of the dye with a mixture of sodium formate/sodium
hydroxide at about 220°C causes the release of p-phenylenediamine
by sublimation (from both structural types). The diamine vapor re-
acts with aniline in an oxidizer solution to form a color. Minimum
quantities of sample required are 5-10 yg.
Mecke, Jr. and Schmahl (1957) reported ultraviolet absorption
maxima for over two dozen aromatic azo compounds.
Earley and Ma (1960) expounded upon the Ti(III) titration of azo
compounds, its applicability in the presence of nitro compounds, and
alterations in the standard technique required.
Sawicki et al (1961) developed a spot test for aminoazobenzene
and its N-methyl and ethyl derivatives which involved reaction with
the compound 3-methyl-2-benzothiazolone hydrazone (MBTH). Table 13
-------
Table 13 . SpectropWiomotric Determination of 4-Aminoazobenzene Deriva-
tives and 'Azotenzene Analogs*
Compotiml
•I-Amiiioazolx'nz^nc
4 At>mm,izol>t:n:-''ne -
A 15
A'-.MfMiyl AH
3'-Mrtli\ i-A -iin-ili vl AB
AM-:tUyl A1J
\-ri-..- u- i AB
'I'-Mclhvltiilo-X-pucnyl
AB
.Y,.\-Diti;-tliyl..!nirionzo-
I>"ti7i'i"- "-•" D Mi"1
S-Jk-thUDVlf
2'-Aniino DAB
2'-Cli'oro DAB
2-r:thyi OAB
2'-Motlio%y DAB
2'-Metliyl DAB
2-!Mi" lli v I-.! '-me t hoxy-
c:'rboi:vl D,\B
2,3 '-Dimethyl OAB
2-Mfthyl-3'~.-hIuro DAB
2-Mcthyl-4'-ncctyl DAB
2-Mc'th\ l-4'-iucthyllhio
D \ I'."
2',5'-DimuUyt DAB
2',4',6'-Tribromo DAB
Ar-Mfit!iyi-A"-etLvSarmnf>-
a7ol.cn.. t.e -"MEAB
2'-Cliloro M CAB
2'-N"itro MKAJJ
3'-Arcta'i;itio MliAU
3-Nitro MKAB
4-Kt'A-l MKAIJ
•I'-l'luoroMEVU
A'-M.-tliyl-.V-liouTiyl AB
A",.V-Hirtli>l AH
o'-Ai'i-t:i»niim DAB
;}'- \jtiiuo DAB
;S'-t'li1oro D \!1
S'-Kllnx.)- DAli
Xro-v«i
Hi ft
Drriv:
.17)
<; i is
5S')
(j.'iO
c»; K
."(Ml
(i'.T):4
tidSj
(,;),">
GC.tis
Cl'l!
OJ I
(*••)}
072
.">' ^
cou
COS
(ji'.'ls
Gi:,'
CliU
6S''>
Cf."u
C05
s?
07 lj
5 VI 3
fiO"»3
5SS
()7(U
5*-.S
f«i">ii
.v»7
6'iGd
5>,"i
ii70^
oo r
C.O.H
C74^
c.m
c.i; ; i
63'M
Cl.'!
(i'lT
GVM
(,(),",
(Jt'.Os
(jl)i)
('.("MS
GDI
007
iilX-,
f.'.IK
('.(III
07 !•<
!'.< l.'i
•iss"
C.7-V
COS
(.li'.S
t,(i ;
(iU'N
« X
10"3
ilU'es
11
5
:>(i
;jl
;;2
3(i
.Tl
;;2
07
5%
39
y
70
;,f,
72
•Jd
-!">
37
7.j
01
07
47
Gt
51
•15
2S
S
71
1!
71
19
65
1,1
71
10
GO
4'1
5
4
ol
42
;>!>
',ii
51 ,
',']'.)
fit!
1".)
01
Kt
4
:;•.*
;;s
"i
70
:!(»
2~>
(.15
*, |
70
r.i
Compound
xmi.
n»M
4-Aniin(ia2obcnzcnc Dcrivatiw
3'-M.-lliyl DAB
3?-.\iIroDAB
3'-Trillnoromctliyt DAB
4'-Ac(.|yl DAB
•I'-Amino DAH
4'-l-:t!ioxy DAB
4'-Ethyl DAB
4 '-Fl uo(0 DAB
4 '-Methyl DAB
4'-SulfoDAB
4'-'L"hiocyano DAB
2,2 '-Dimethyl DAB
cm
CCls
010
(ili.'H
fifl.S
(.(i.'is
000
(JfiOs
.IS.)
CCSa
5^8
C>i>.">s
003
CrfV.M
00:5
«i!s
an
«<;0s
fi)2
fiCGs
ft' 1 9
OfiSs
5(i
4 ;
37
2(i
4-">
.'if
.r>7
•i'l
IS
12
7!
r>
Azobt'nzRiic Analog?
4-l'llu'iiyJrizo-l-ii:>.iihthy!-
insiitie
)-|A",A"-Ditni'(hyl-4-
nminnplioiij l.-izo]-
Tl'lph(tl:ll(.'lU!
2-[.V, A"-Dimi'fIivI-l-
aminophruj I'i?oj-
/Icorono
Reprinted with
Anal. Chem. , 33
by the American
SS7
COS
OG2s
573
CSOs
40
6d
55
12
4
lttj;j__
permission from
: 1574-9 (1961). Copyright
Chemical Society.
•* \ll v:ilu<"* In .<•>( mi minimi mi uf tun i!*leruu!i i
* Mo! ir .il<5t
-------
gives the compounds which react with MBTH. These compounds did not
react completely: 4'-acetyl-N,N-dimethylaminoazobenzene (4'-acetyl-DAB),
4'-methylmercapto-N-phenylaminoazobenzene, 2',4',6'-tribromo-DAB, 4'-
thiocyano-DAB, and 2'-methoxycarbonyl-2-methyl-DAB. These compounds
did not react al all: azobenzene, 4-hydroxyazobenzene, 4-methylmer-
captoazobenzene, and 4'-nitro-N,N-diethylaminoazobenzene. These com-
pounds gave a reaction, but the product had a molar absorptivity less
than 4,000: 4'-nitro-N-phenylaminoazobenzene, 4'-?henyl-DAB, 4'-nitro-
N-methyl-N-ethylaminoazobenzene, 3-methyl-DAB and N,N-dimethyl-p-(p-2-
tolylazo-2-tolylazo)aniline.
Villanua et al (1962) presented in tabular and graphic form the
UV spectra in acidic, alcoholic, and alkaline solution of the banned-
for-food water insoluble azo dyes: Methyl Red, Sudan I, II, III, and
IV, Orange SS, Yellow AB and OB, o-aminoazotoluene, and 4-dimethylamino-
azobenzene. Rf values for circular paper chromatography were also given.
Chikryzova and Podolenko (1964) presented a mettiod suitable for
monitoring the concentration of a known dye. It involved reduction at
a Hg cathode, and titration of iodine liberated at the anode. Inter-
ference from oxygen traces did not become bothersome until the dye
concentration dropped below 1 yM.
Bowie et al (1967) presented in tabular form the mass spectra of a
wide variety of substituted azobenzenes.
Venturini (1967) detected acidic azo dyes in wine by adsorption on
polyvinylpyrrolidinone.
Gemzova and Gasparic (1967) analyzed disperse azo dyes by reductive
cleavage of the azo linkage with Zn in hot acetic acid, followed by
-------
paper or thin-layer chromatography of the released amines. Rf values
for many amines were given, along with those for DNP-hydrazones of
diamines or amino-phenols which were first oxidized to quinones.
01 and Inaba (1967) used infrared spectroscopy to identify amaranth,
New Cocaine, Orange 1, Ponceau SX» and Sunset Yellow. In order to use
NaCl cells, aqueous solutions of the dyes were extracted with 5% Amber-
lite LA-2 (a high molecular weight amine) in CS2- Characteristic ab-
sorption bands were given.
Manukian and Mangini (1971)» by various spectroscopic and direct
synthetic means, identified the dye Colour Index Pigment Red 178 as
the reaction product of two molecules of p-aminoazobenzene with perylene-
tetracarboxylic acid dianhydride, the amine N's having displaced the
ether O's of the anhydrides.
B. Separation-Analysis
Edwards, Jr. et al (1956) reported R values on various adsorbents
using one or more eluants for p-hydroxyazobenzene*, p,p'-azophenol*,
p,p'-azodimethylaniline*, N,N-dimethyl-p-phenylazoaniline, azobenzene,
p-chloroazobenzene, p-methylazobenzene, and p-phenylazobenzoic acid
(melting points given for compounds with asterisk).
Fukui et al (1956) studied the paper chromatographic separation of
p-amino-, p-methylamino, and p-dimethylaminoazoben^ene using water mixed
with a variety of alcohols, ethers, acetone, methylnitrile, and amines;
Rf values and judgments as to suitability of the various eluants were
given.
Ward et al (1959) chromatographed a variety of azo compounds pre-
pared from diazotized p-nitroaniline or beta-naphthylamine with alpha-
-------
naphthylamine, beta-naphthylamine, 5- and 8-nitro-alpha-napht.hylamine.
Ultraviolet absorption maxima, minima, and log e's, along with some
melting points were tabulated.
Fore and Walker (196?) used a combination of papur and thin layer
chromatography, and synthesis to determine the composition of the
(foreign) food dye Brown FK. The two major components were 2,4-diamino-
5-(p-sulfophenylazo)toluene(I) and l,3-diamino-4,6-bis-(p-sulfophenylazo)~
benzene(III). In lesser quantity was 1,3-diamino-4-(p-sulfophenylazo)-
benzene(II). In still lower amounts were 2,4-diamino~3»5-bis(p-sulfo-
phenylazo)toluene(IV) and l,3-diamino-2,4,6-tris(p-sulfophenjrlazo)-
benzene(VI), A trace of l,3-diamino-2,4-bis(p-sulfophenylazo)benzene(V)
was found. In addition two unidentified, colorless components were found.
The two major components did not result from the dye synthesis as acci-
dental byproducts, but were intentionally formed. Some evidence was
presented for the necessity of some of the minor components in the
successful use of the dye. Figure 2 presents the structures of the
identified components.
NH
Reprinted with permission from Food
Cosmet. Toxicol. 5:1-9 (1967). Copyright
by the Pergamon Press Ltd.
NH,
NH,
Y « s
F»0. 2 . Structurei and synthetic pathwayj Of Brown FK component).
-------
Brain et al (1971) chromatographed amaranth and Sunset Yellow FCF
following British Standards. Depending upon the manufacturer the main
peak in amaranth represented 78.4-99.7% of the whole, the impurities,
including the azo dye Fast Red E, also showing vajiations. In the case
of FCF the major component comprised 93.6-98.6% of the whole, but the
impurities showed a greater range. The impurities seemed to be charac-
teristic of a particular manufacturer and could be used to identify him.
Maraion (1972) described an AOAC method of analyzing FD&C Yellow No.
6 for traces of the materials from which it is synthesized: sulfanilic
acid (diazotized) and 6-hydroxy-2-naphthalenesulfonic acid (Schaeffer's
salt), and for an impurity in the latter, 6,6'-oxybis(2-naphthalenesul-
f onic acid). The method involved column chromatography over cellulose
using 20 and 40% aqueous diammonium sulfate as eluar.t, followed by UV
analysis of the eluates. The impurities eluted in the order in which
they were mentioned above.
C. Separation
Birnbaum et al (1953) separated the cis and trans isomers of azo-
benzene and derivatives by irradiating a petroleum ether solution with
a Hg arc and filtering over an alumina column which retained only the
cis. They obtained UV spectra for the trans and stable cis isomers.
Melting points were given for the isomer pair of the para derivatives:
iodo, bromo, chloro, fluoro, ethoxy, methyl, carborethoxy, nitro,
carboxy, cyano, dimethylamino, and p,p'-dimethyl.
Frankel and Wolovsky (1954) separated the cis and trans isomers
of azobenzene by paper chromatography using the eluant 40% acetic acid.
U9
-------
Silk (1963) presented a column chromatographic method, on Celite,
for separating lipstick dyes. '
Topham and Westrop (1964) found that thin layer chromatography on
silica gel G, developed with 95/5 chloroform/meth mol, provided an ade-
quate separation of: 4-aminoazobenzene (AB), 4'-bydroxy AB, N-methyl AB,
4'-hydroxy-N-methyl AB, N,N-dimethyl AB, and N,N-dimethyl-4'--hydroxy AB.
An appropriate range for detection in a mixture was 25 ng-1 ug.
Gurevich and Chukreeva (1967) presented Rf values on four activity
grades of silicic acid for azobenzene, p-aminoazobt:nzene, p-hydroxya zo-
benzene, p-methoxyazobenzene, Sudan yellow, and Sudan red. Any of the
grades seemed suitable for the separation of a mixture of all six using
carbon tetrachloride.
Parrish (1968) found that Sephadex G-25 was suitable for the separa-
tion of azo dyes. Judicious adjustment of the ionic strength of the
water used as eluant provided a means to alter Rf values, and thus
separate complex mixtures using more than one column or pass.
Nairay et al (1969) reported Rf values on silica gel for the cis
and trans forms of 4-amino-4'-ethylazobenzene with 0, 1, or 2 methyl
groups on the amine, and 2 or 4 fluoro atoms on the amino phenyl ring.
Considering the precautions taken to isolate these geometrical isomers,
it appears that most previous publications reporting Rf values dealt
with mixtures.
Hall and Perkins (1971) disclosed a procedure for purifying commer-
cial dyes of isomers and color standardization adjuvants. Essentially it
consisted of extracting either an aqueous solution of the dye with acidi-
fied butanol, or the undissolved dye with hot N,N-dimethylformamide in
-------
cases of butanol insoluble dyes. Impurities or additives remained behind
or were selectively retained in the extractant while the dye was re-
precipitated.
Gasparic (1972) discussed paper and thin layer chromatography of
azo pigments and lakes. Warm N,N-dimethylformamide was the best sol-
vent for spotting these sparingly soluble substances, but it had to be
thoroughly removed before proceeding with development by benzene or
toluene. More polar developers were required for the lakes.
Gilhooley et al (1972) described procedures for extracting food
dyes from water soluble foods, baked goods, and processed meats. The
extracts were then chromatographed over polyamide columns to further
purify the dyes, prior to identification by known thin layer chromato-
graphic techniques.
-------
VII. CHEMICAL REACTIVITY
A. Environmental and use associated reactions
In the application of dyes to whatever is intended to be colored,
chemical reactions are sometimes employed but they do not involve the
azo linkage. The amphoteric protein nature of wool and silk renders them
easily colored by dyes having amine or sulfonic acid groups through salt
formation. Dyes containing these groups and/or hydroxy or carboxy groups
can be fixed to cotton by a process called mordanting. This involves
first reacting the cotton with a metal oxide (for acid groups) or tannic
acid/tartar emetic (for basic groups). Again, simple acid-base reactions
are involved. Generation of the azo group, and thusly the dye, on cotton
itself has been practiced nearly a hundred years. Ir "ice coloring" a
phenol is soaked into the cotton, and then reacted with an iced diazonium
solution. In "ingrain dyeing" an amine is first applied to the cotton,
then diazotized, and finally immersed in a phenol solution. A dye type for
cellulosics that covalently binds to the fiber has been developed in the
last 15-20 years, and consists usually of a suitable dye which has been
modified by the addition of a dichlorotriazinyl group. The dyeing takes
place in an alkaline medium to assist the displacement of one of the chlor-
ine atoms by one of the hydroxyl oxygen atoms of the fiber. Alternatives
to the dichlorotriazinyl group are vinylsulfonyl (-SC^-CH = CH2) an^ ac~
tivated alkyl hydrogen sulfate; both give ether linkages with the fiber
-------
in the same fashion as the triazinyl.
When used as chemical foaming agents the two compounds azodicarbonamide,
H2NC(0)N = NC(0)NH2, and azobisisobutyronitrile (AIBN), [NCC(CH3)2N=]2, decom-
pose when heated to give off nitrogen gas. The residual parts of the
molecules may decompose further or combine with one another. When the AIBN
is used to initiate polymerization, it breaks apart into NCC(CH3)2N' radi-
cals. These eventually recombine, combine with H- radicals, or other
radicals generated in the overall process or present on equipment walls.
Mytelka and Manganelli (1968) demonstrated that irradiation with a
Co-60 source of the mother liquor from production of Direct Red 79,
assisted by oxygenation, decolorized the liquid and reduced its oxygen
demand. Furthermore, the treated waste became more biodegradable.
Trimmer (1971) reviewed the recent literature and also reported the
results of his own studies on purifying textile plant wastes of dyes.
A variety of oxidative methods, among some non-chemical ones, were tried
but it did not look as if any one treatment method could be considered
generally applicable.
Evans (1971) indicated that azobenzene, in an acidic, dilute ethanol
solution, was converted by sunlight via the Beckmann rearrangement into
benzidine (probably far more toxic than azobenzene) and via intramolecu-
lar ring closure into benzo[c]cinnoline (9 ,10-diazapher.anthrene) .
Van Beek et al (1971) in a study of 18 azo dyes in water or ethanol
solution found that flash photolysis in the presence of a proton donor
first produced -NH-N- radicals. Two of these dispropoitionated to form
-NH-NH- and -N=N-. The -NH-NH- decomposed to -NH2 or reverted to -N=N-.
B. Aspects with biological implications
Cilento (1952) found that o-aminoazotoluene, p-diethylaminoazobenzene,
-------
and 3'-methyl-dimethylaminoazobenzene formed complexes with bile acids.
von Euler et al (1952) heated p-dimethylaminoazobsnzene with
cysteine'HCl in methanol. They recovered 1/4 of the dye unchanged, and
1/4 as 1,4-diaminobenzene. In another experiment incubation of the dye
with ground rat liver for two hours at 37°C resulted in complete loss of
color.
Kawai (1952) studied the decomposition of p-aminoazobeuzene, p-
methylaminoazobenzene, and o-aminoazotoluene by fresh slices of rat liver
or kidney. The N-methylated dye decomposed at a slower rate than the
other two. The liver showed the greater capacity for decomposition.
Supplementation of the basic rice diet with yeast, li.ver powder, or
liver extract improved the ability of liver slices tc decompose the dyes.
Spiking of the slices with riboflavin likewise increased this.
Diemair and Boekhoff (1953) studied the inhibition of pepsin (in
gastric juice) by azo dyes. The order of decreasing inhibition found
was: Orange GG, Brilliant Black = Naphthol Red S, Ponceau 6 R, Fast
Yellow Extra, Bordeaux R = Orange SXX = Tartrazine XX, Cochineal Red A =
Yellow 27175, Fast Red E, Thia^ine Brown R. The concentration range
50-1000 mg/1 was used, the Orange GG showing 3% inhibition at the low eni^
and the Thiazine Brown R only 13% at the high end. Total inhibition at
the high end was found with the first four dyes mentioned. In a follow
up study on inhibition of trypsin, the dyes Bordeaux F, Brillian Black,
and Orange SXX were found effective.
Burkhard et al (1953) found that the binding of albumin from bovine
serum to 4-amino-4'-sulfoazobenzene was slight, but increased if the
amino group was removed or alkylated.
von Euler et al (1954) reported on the metabolism uf p-dimethyl-
-------
aminoazobenzene by rats. When incubated for 24 hours with 1 g of normal
rat liver, as much as 40 yg of dye was totally used up. When cancerous
liver was used under the same conditions, 0, 55, and 75% of 10, 20, and
40 ug quantities of dye, respectively, remained. Homogenized intestine
in 24 hours used up 99-100% of the dye. There1 appeared to be some limit
on the ability of the rat to clear the dye rrom its stomach as 2.3 mg
remained 12 hours after introduction of 12 mg; J or comparison, 0 rag
remained 23 hours after dosing with 18 mg. Jilood concentration of azo
dyes, 6.4 vg/g> was found 20 hours following an oral dose of 12 mg of
the dye at the conclusion of a 55 day period during which 540 mg of
dye was administered, orally.' The liver contained 0.35 Ug/g of a mix-
ture of the dye and its mono- and di-demethylated metabolites, at the
end of an eight month period of daily oral dosing with 6 mg dye.
Hatem (1959) reported that histamine formed complexes with the
carcinogens: p-aminoazobenzene, 2',3~dimethyl-4-aminoazobenzene, and
3'-methyl~4-dimethyluminonzobenzene.
Kusama ( i4tiO) introduced Into the stomachs of male rats the carcino-
genic azo r.oii,(jotinds : p--N,N-dimethyia«iinoazobenzene , 2 ' ,3-dirnethy 1-4-
aminoazobenzeiK-, p-aminoazobenzeae , p-rJ-methylaiiunoazobenzene , and 3'-
methyl-4-N ,N- d i me thy ! aiiiinoazobenzerie . He then showed that liie dye.s were
bound to tvrushie mo 1 Denies in the ]ivi-r.
Matsumoiu and Teidy-una (1961) compared t.!ir> rates of N-demothvlation
by rat liver homogenate oi various N- and ring-methyl-subs r j tut ed p-
methyl, alkylaminoazobenzenes. They did not I ind any correlation between
the rates and the known carcinogen!cities.
Matsumoto (1961) used homogenized rat liver to N-demethylate a
variety of C- and N-alkylated p-aminoazobenzenes. It was found that those
-------
compounds which resisted demethylation were also non-carcinogens;
however, only some of those which did demethylate were carcinogens.
Ishidate and Hanaki (1961) conducted non-enzymi". oxidative N-
demethylations of ring methylated p-N,N-dimethylaminoazobenzene. They
found that the reaction rates correlated with the pi-electron density at
the amino N, but not with carcinogenicity.
Callander and Roberts (1961) studied the ability of hydrogenase
from Azotobacter vinelandii and Desulfovibrio desulfuricans to catalyze
the reduction of azo linkages. The compounds tested and the results are
given in Table 14. A + means that reduction occurred. A + in the
Indirect column means that a "carrier", benzyl viologen (from 0.5-1 part
per 1000 parts of azo compound), was required for reduction to occur.
Seemingly, direct reduction was related to the ability of the groups
bonded to the azo linkage to withdraw electrons from It.
Sikorska and Krauze (1962) studied the effects o± FD&C dyes on the
activity of succinic oxidase from rat liver (homogenized). At the level
of 4 yg dye/mg rat liver only food Black No. 1 showed any effect, an
8-16% inhibition. At 400 yg/mg: chrysoidine had no effect; food Yellow
Nos. 3 and 4 had an inhibition of <20%; direct Blue No. 5, food Red Nos.
2, 3, and 7 had an inhibition of 20-50%.
Manchon et al (1962) studied the effect of methyl orange on oxygen
consumption of various substrates and rat liver homogenate or a supernatant
thereof. Using homogenate and glucose-6-phosphate (3.3 mM) there was a
17% decrease when the concentration of dye was 14-86 yM; using supernatant
the decrease was 40%. On a different strain of rats these figures were
11 and 32%, respectively. Using homogenate, 10 mM oc-ketoglutarate, citrate,
or succinate, and 0->195 yM dye, there was a 36% decrease for the ketoglu-
-------
Table 14. Reduction of Azo Bonds by Hydrogenase
THE CONCENTRATION OF SUBSTKATK WAS IO~* M IN ALL CASES
CH,-N^N- CH,1' —
(CHj)2ClI- -N=K-CH(C1I,}," —
(trans)* —
V X=L\-/~V POj X a *
/~v V.V.^T-X
c<> ,Ka *
70% inhibition of enzyme at a
concentration of lo~2 Af-
o
+
Stimulated by Fc:+, Cu2 ^, Co!+,
\=N
Slow rate of reduction (10% of rate of
reduction of trans)
Rate of reduction same fo 2,2'-isomcr,
No metal stimulation
CH,
CH,
CH,
/
CH3
CH,
CH,
Low rate of reduction: approx. 5 % of rate of
reduction of 2,j'-azopyridme
Rapid reduction; approx. 20 x that
of z.z'-azopyndine
-------
tarate (mostly reached at about 60 yM dye), nominal decrease for the
citrate, and a 53% increase for the succinate (still rapidly increasing
at 195 yM). With the substrate D-phenylalanine (20 mM) and 86 yM dye,
a noticeable decrease in oxygen consumption occurred.
Manchon and Lowy (1962, pp. 1619-22) demonstrated that the ability
of rat liver homogenate supernatant to reduce the azo linkage of methyl
orange was increased when the rat had been allowed to drink water con-
taining 0.2 g/1 of the dye for 18-39 days. However, this higher activity
supernatant was inactive against ethyl orange.
Matsumoto and Terayama (1965) compared the rates of reduction by
rat liver hydrogenate of the azo linkage of ring-methylated mono- and
dimethylaminoazobenzenes, and also p-R2N-azobenzene (where R2 is all
possible combinations of H, Me, and Et). They found nc correlation
between the reduction rate and carcinogenicity, or pi-electron density
at the azo linkage.
Matsumoto and Terayama (1965, pp. 331-7) gave rats oral doses of
various derivatives of p-aminoazobenzene. From the livers they ex-
tracted dyes indicative of N-dimethyl types breaking down to N-methyl
and -NH2 , and of N-methyl types breaking down to -NH2 or dispropor-
tionating to -NH2 and N-dimethyl. Those dyes having -NH.2 to start and
methyl groups on one of the phenyl rings were not able to produce N-
methyls or N-dimethyls.
Matsumoto and Terayama (1965, pp. 339-51) found that one of the
liver metabolites from feeding 2' ,3-dimethyl-4-aminoai,obenzene to rats
was 4,4'-bis(o-tolylazo)-2,2'-dimethylazobenzene, apparently resultant
' from oxidative coupling of the 4-amino group of the parent (also known
as o-aminoazotoluene).
-------
Higashinakagawa et al (1966) studied the degradation in rat liver
of orally administered N,N-dimethylaminoazobenzene. Apparently the firsr
step was loss of one of the methyls. Then the remaining methyl became
coupled to the sulfur atom of a methionine residue, thereby generating
the precursor to the polar dye commonly extracted from the rat liver
after chemical decoupling of the methionine from its protein tail.
Westrop and Topham (1966) found as liver metabolites in rats fed
4-dimethylaminoazobenzene: 4-methylaminoazobenzene, 4-aminoazobenzene,
4'-hydroxy-4-dimethylaminoazobenzene, 41-hydroxy-4-methylaminoazoberzene,
4'-hydroxy-4-arninoazobenzene, and 4 '-hydroxy-4-acetylaminoazobenzene.
The same four hydroxylated metabolites were found when 4'-fluoro-4-
dimethylaminoazobenzene was used, but in lower amounts. This ready dis-
placement of the fluoro atom was unexpected, and it raised some concern
about the validity of studies which had introduced fluorine atoms at
various positions on the phenyl rings of azobenzenes to determine if
these positions were actively involved in carcinogenesis.
Westrop and Topham (1966, pp. 1395-9) found that there was uo
correlation between carcinogenic!ty and total 4'-hydroxylated metabolites
in the liver of rats fed 3-methyl-4-methylaminoazobenzene,4-dimethyl-
aminoazobenzene , and the latter's 2-,2'-, or S'-methyl derivatives. In
contrast there was a correlation with these 4'-substituted derivatives:
chloro, ethyl, fluoro, methoxy, methyl, nitro, and trifluoromethyl. The
authors hypothesized that the hydroxylation occurred initially on the
amine N after one demethylation, followed by an internal rearrangement
of the hydroxy to the 4' position. It had not been possible to prepare
the intermediate N-hydroxy to test this.
Turba et al (1966) incubated homogenized rat liver with 3'-methy 1-
-------
N,N-dimethyl-4-aminoazobenzene which had a C-14/H-3 ratio of 0.60.
This ratio was 0.43 and 0.62 in the cytoplasmic and rdcrosomaL protein
fractions, respectively, of the post-incubation homojjenate. This differ-
ence was interpreted as indicative of N-demethylation occurring while the
dye was bound to the cytoplasmic protein.
Daniel (1967) examined the comparative rates of azo linkage reduc-
tion of tartrazine, Orange II, and Orange G by rat liver homogenate
supernatant, and found them to be 1, 2.5, and 3.4, respectively (male
rats), or 1, 2.5, and 2.9, respectively (female rats). The difference
between male and female rats in the reduction of Orange G was deemed
of low significance because of wide variance.
Lolua et al (1967) commented that iron was capable of reducing azo
linkages in acid media to the hydrazo (-NH-NH-) or amine stage. They
found that 0.1% of sorbic acid was sufficient to protect amaranth,
chrysoidine, and tropaeolin.
Ryan et al (1968) tested the ability of rat liver homogenate and
protein preparations from the intestinal bacteria E. coli and Proteus
vulgaris to reduce the azo linkage of a variety of water-soluble azo
dyes. Their results are presented in Table 15, and are indicative of
metabolism of common food and drug dyes occurring in the intestine, not
the liver.
-------
Reprinted with permission from Nature
219(5156):854-5 (1968). Copyright by
MacMillan Journals Ltd.
Table 15. Reduction of water-soluble azo dyes by rat liver homogenate
supernatant and soluble bacterial preparations
Percentage reduction*
Color Bacterial
Dye Index No. Liver Proteus E. coli
Tartrazine 19140 4.0 54 24
Lissamine fast 18965 17.0
yellow 2G
Amaranth 16185 76 95 91
Ponceau SX 14700 0 fc5 81
Fast yellow 13015 41 95 91
Naphthalene fast 16230 9.0 95 92
orange 2G
Sunset yellow 15985 11.0 95 95
m-Methyl orange — 72 93 60
Neoprontosil — — 95 60
* Incubated anaerobically, and assayed after 60 min. Corrected for
protein binding.
The incubation medium contained liver homogenate equivalent to 250 mg
wet weight, or soluble bacterial protein (2 mg) together with dye (1 pM),
MgCl2 (2 yiM) , NADP (300 yiM) , glucose-6-phosphate (250 yM) , glucose-6-
phosphate dehydrogenase (1 Kornberg unit) in 0.07 M phosphate buffer,
pH 7.4.
Maher et al (1968) allowed N-benzoyloxy-N-methyl-4-aminoazobenzene
and biologically active DNA from Bacillus subtilis tc interact at room
temperature and pH 7.5. As a result the DNA suffered severe reduction
in transforming activity, increase in frequency of mutation, and decrease
in buoyant density. None of these changes resulted from contact of the
DNA with 4-methylaminoazobenzene.
Lin et al (1968) identified the polar dye P2b from the liver of
rats fed 4-methylaminoazobenzene as 3-(homocystein-S-vl)-4-methylamino-
azobenzene .
Wu and Smuckler (1968) isolated rat liver microsomes and incubated
them with 4-amino-, 4-methylamino-, and 4-dimethylaminoazobenzene.
Cleavage of the azobond was much faster in the monomethylated compound.
Demethylation rate was equivalent. The optimal cleavage conditions did
61
-------
not produce any demethylation. Rate of cleavage was always greater than
rate of demethylation, apparently just the opposite of the situation in
intact animals.
Lin et al (1969) reacted tyrosine with N-benzoyloxy-N-methyl-4-
aminoazobenzene to give a pair of polar dyes. These were shewn to be
identical (spectroscopically, chromatographically, aiid chemically) with the
two polar dyes, commonly designated Pla and Plb, which may be. isolated
from the liver of rats fed N-methyl-4-aminoazobenzene. Pla was tempor-
arily described as N-(3-tyrosyl)-N-methyl-4-aminoazobenzene, and Plb as
3-(3-tyrosyl)-N-methyl-4-aminoazobenzene. By oxidation with hydrogen
peroxide the authors were able to convert synthetic 3-(homocystein-S-yl)-
N-methyl-4-aminoazobenzene into a sulfoxide which was identical with the
minor polar dye Pic. These four dyes Pla, Plb, Pic, and P2b comprise
90% of the polar dyes derived from hepatic protein-bound dyes by suc-
cessive enzymic and hot alkaline hydrolysis.
Matsumoto and Terayama (1970) gave rats p-methyl-, and dimethyl-,
and methyl ethyl aminoazobenzenes in which the carbor.s and/or the protons
on the carbons of the alkyl groups were radioisotopes. Analysis of the
polar dyes isolated from the liver indicated that: the ethyl group was
preferentially cleaved, only one methyl of the dimethyl compound was
cleaved, and the dye was not bonded to the liver protein through the
N-methyl group (the latter in confirmation of work by Lin et al (1967).
Harris et al (1971) showed that Me2NC(0)N = NC(0)NMe2, a tetra-
methylated azodicarbonamide, reversibly converted glutathione (GSH) to
the oxidized form (GSSG) in nucleated mammalian cells with only nominal
oxidation of protein SH. The authors considered the azo compound a use-
ful tool in the study of the biochemistry of GSH.
-------
Deleted because of copyright clearance
Gangolli et al (1972) demonstrated that the complexes between rat
serum protein and the azo food dyes Sunset Yellow FCF, Black PN, and
Black 7984 readily separated during electrophoresis on cellulose acetate.
-------
The protein-amaranth complex did not separate on cellulose acetate, but
did on poly aery land, de gel.
DuPlooy and Dijkstra (1972) studied the polar extractable metabolites
from the liver of rats given 4-dimethylaminoazobenzene. Between one and
thirteen hours after dosage six metabolites were present at any one time,
but at the fourth hour the amount peaked and consisted mainly of the
0-sulfate esters of 4'-hydroxy-4-methylaminoazobenzene and 4'-hydroxy-4-
dimethylaminoazobenzene. If at the time of dosing an i.p. dose of
methionine-S-35, or if 1/4-1/2 hour before sacrifice a s.c. dose of ionic
sulfate-S-35, were given, then S-35 was incorporated into the metabolites.
Henkens and Sturtevant (1972) reported that the esterase activity
of bovine carbonic anhydrase was completely inhibited by the metal-chelator
4-(8-hydroxy-5-quinolylazo)-l-naphthalenesulfonate.
Connors et al (1972) compared the ability of various preparations
from rats to reductively cleave the azo bond of 2'-cavboxy-4-di(2-
chloroethyl)amino-2-methylazobenzene. The preparations were supernatants
from homogenates of gut, gut (entire homogenate), Walker 256 tumor cells,
spleen, kidney, and liver. The relative rates of cleavage, in the same
order, were: 1, 4.4, 5.3, 7.9, 8.1, and 27.
Albrecht et al (1973) compared the effects on liver functions of
medium term feeding to rats of amaranth and 4-dimethylaminoazobenzene.
The animals were fed from weaning, up to 2, 3, 4, and 9 months of age,
consuming over these periods (in grams): 1.4 or 0.6, 3 or 1.2, 16 or
1.9, 43 or 5.1 of amaranth or the carcinogen, respectively. The amaranth
had no effect on weight gain or liver weight (as a percentage of body
weight). The carcinogen caused a noticeably lower weight gain at 2
months, but at 9 months the controls were only slightly heavier. The
6k
-------
carcinogen caused liver weights to be much higher as a percentage of body
weight. The amaranth had no effect on the percentage of protein in the
liver, while the carcinogen caused reductions at 2 and 9, but not 3 and 4
months. The amaranth had no effect on glucose-6-phcsphatase, while the
carcinogen lowered it at 2, 4, and 9 months. The amaranth had no effect
on glucose-6-phosphate dehydrogenase, while the carcinogen raised it at
2 and 9 months. The amaranth had no effect on 6-phosphogluconate dehydro-
genase until 9 months, when a lowering occurred; the carcinogen, on the
other hand, raised this enzyme level at 2 and 9 months. Amaranth had no
effect on the ability to cleave the azo bond of amaranth, while the car-
cinogen increased this ability at 2, 4, and 9 months (on a per 100 mg of
protein basis). Neither dye had any effect on the activity of NADPH-
cyto chrome C reductase. Liver homogenate supernatant was used for the
enzyme studies.
Chauveau and Benoit (1973) fed weaned rats a diet containing 0.06%
4-dimethylaminoazobenzene (DAB) or 0.063% 2-methyl-4-dimethylaminoazo-
benzene (2-Me-DAB) for 1-3 weeks. DAB bound to total liver protein was
the same at 14 as 7 days, then increased 20% at 21 days. Contrastingly,
2-Me-DAB bound to total liver protein was the same as DAB at 7 days, but
increased steadily (by 57% at 21 days). DAB bound to liver DNA peaked
at 14 days, then dropped by more than 50% at 21 days. The 2-Me-DAB
bound to liver DNA peaked at 7 days and remained unchanged. The DNA-
bound DAB/2-Me-DAB ratio was 2.47, 3.69, and 1.6 at 7, 14, and 21 days,
respectively.
-------
VIII. BIOLOGY
A. Metabolic effects
1. absorption
Radomski and Mellinger (1962) found only 2-4% absorption by rats of
the food dyes amaranth, Ponceau SX, and Sunset Yellow from the g.i.tract.
Ryan and Welling (1967) found that chemically pure Sudan III fed to
rats as a suspension in methyl cellulose mucilage, olive oil, or oleic
acid showed negligible absorption from the g.i. tract. Previous reports
to the contrary presumably resulted from the use of impure commercial
material.
Walker (1970) reviewed the literature on metabolism of azo compounds,
including absorption. His review indicates that in general, highly sufon-
ated dyes aren't absorbed. However, there is a good pjssiblity that they
may be cleaved at the azo bond by the intestinal flora, and the metabol-
ites may be absorbed. The same "pre-metabolism" interferes with judgments
on the extent of absorption of the non-sulfonated, oil soluble dyes.
Gibaldi and Grundhofer (1972) found that the permeability of everted
rat small intestine to methyl orange increased 100% after a 1/2 hour con-
tact period.
2. excretion
MacDonald et al (1953) studied the metabolism of the N-methyl groups
of strongly and weakly carcinogenic members of the N- and ring-methylated-
4-aminoazobenzene series. Using C-14 labeled N-methyl groups for ease of
detection, they reported the results in Table 16. Prefeeding of the
appropriate dye (non-labeled) for three weeks prior to the gastric tube
dosing with the labeled sample (+ in the second column) had no consistent
effects on the excretory pattern. No discernible pattern correlating
66
-------
TA11LE 16.
DISTRIBUTION OF O IN THE HESPIRATOIIY CAiinox DIOXIDE, UUINK, AND FKCES OF
RATS FED CERTAIN N-Mi;Tnyi,-Ci<-LAiti;tKO DYKS
Pen CENT or TOTAL ACTIVITY ADWINIRT^KF.D
Arrontit^l
N-MnnvirC"- rH».n;ramo Expired COi Kccti l.ridf lot in
t,A l,eo or urJUAUKI'KD
1»K DTK 5 hr. 10 hr. 48 hr. 48 hr. 4 18 71
+ 81 40 51 4 18 74
3'-Mctliyl-DAB - 2* 47 66 7 14 87
+ 23 42 50 5 81 8*
4'-MetI>yl-l>AB - 21 35 +6 H I'l 77
+ «0 ,'!3 47 0 hi (iO
4'-Mctliyl-MAB - 23 42 60 7 2.) Of)
+ 22 37 49 8 35 02
S-Methyl-MAH - 24 40 60 6 lO 76
+ 20 38 59 0 H 82
Reprinted with permission from Cancer
Research 13:292-97 (1953). Copyright hy
Cancer Research Inc., and the Americar.
Associatjori_for_Cancer Research.
carcinogenicity or degree of N-methylation with distribution of excreted
C-14 was found.
Ishidate and Hashimoto (1959) examined the urine of dogs after oral
dosing with 4-aminoazobenzene (AB), 4—dimethylaminoazobenzene (DAB), or
4'-hydroxy-4-aminoazobenzene. In the 3-7 hour urine was found AB, N-methyl
AB, 3-KOS03-AB, 4'-KOS03-AB, 3-HOS03-AB, 3-HOS03-N-glrcosiduronate-AB
(probably). In the 24 hour urine was found o- and p-hydroxyaniline,
toluene, p-phenylenediamine, AB, 4-methyl AB; the o-hydroxyaniline pre-
dominated.
Ryan and Wright (1961) examined the biliary excretion of unchanged
dye after i.v. injection of a number of food quality dyes in rats. Their
results are given in Table 16a, and do not indicate any relationship between
structural type and metabolization.
67
-------
Reprinted with permission from J. Pharm.
13(8): 492-95 (1961). CopyrighlTBy
Masson ET CIE
TABLE I6a.
BILIARY EXCRETION OF \VATER SOLUBLE SULPHONATED AZO DYES FROM RATS
Nune
Axohrn:rnfs
3 -Su!pho-4-
-------
27 day period 86% of excreted activity was in the feces, 14% in the urine.
About 43% of the administered dose of activity appeared in the feces within
24 hours, then fell off rapidly. Urinary activity was about the same at
27 days as at 72 hours. There were three more colored metabolites of No.
14 in the urine than No. 2 (2), and a total of nine suspected compounds,
the only identification being l-amino-2-naphthyl sulfate.
The intestinal contents of five rats were removed and mixed with
200-300 ug/g of No, 2. Within 24 hours there was no remaining intact dye
in two cases, all of it remaining in two cases, and 23% remaining in the
last. The same experiment on dog and rabbit intestines showed rapid dye
destruction in all cases. Attempts to measure the fecal excretion of ad-
ministered dye and then run the in vitro study on the intestine of the
same rat showed no correlation between in vivo and in vitro results. The
in vitro rat, rabbit, and dog studies with No. 14 gave the same results as
with No. 2.
Radomski (1962) in a follow-up study was able to confirm the presence
of the 0-glucuronide of l-amino-2-naphthol in the urine of rats given ex-
ternal D&C Red No. 14. He was not able to detect thir substance in the
urine of dogs similarly dosed.
Radomski and Mellinger (1962) found that in oral dosing of rats with
amaranth, Ponceau SX, and Sunset Yellow food dyes, increased quantities of
unchanged dye in the feces could be generated by dosing with antibiotics
to depress the activity of the intestinal flora. Of the 2-4% of these dyes
which was absorbed through a normal g.i. tract, most was excreted unchanged
in the bile.
Robinson et al (1964) gave rats i.p. injections of 4-aminoazobenzene
(AB), N.N-dimethyl AB, 3',N,N-trimethyl AB, or 2,N,N-trimethyl AB, and
69
-------
then analyzed the urine at 24, 48, and 72 hours for certain azc cleavage
metabolites (the urine was acid hydrolyzed). About 10% of the N,N-dimethyi
AB appeared as p-phenylenediamine, and 70% as p-aminophenol within 24
hours; neither increased over the additional 48 hours studied. About 18%
of the 3',N,N-trimethyl AB appeared as p-phenylenediamine, and 28% as 4-
amino-2-methylphenol within 24 hours; neither increased over the additional
48 hours. About 18% of the AB appeared as p-phenylenediamine, and 90% as
p-aminophenol within 24 hours; neither increased over the additional 48
hours. Only the 2,N,N-trimethyl AB showed increases with time; one
metabolite, 2,5-diaminotoluene rose" from 14 to 37 to 78%, while the other
metabolite, p-aminophenol, rose from 47 to 73 to 80% of the theoretical.
The separate injection of p-phenylenediamirie resulted in enough more of
it being excreted in the urine to indicate that the low amounts found
above didn't likely result from in situ destruction.
Radomski and Harrow (1966) administered l-(o-tolylazo)-2-naphthylamine
(Yellow OB) into the stomachs (ligated just beyond the pyloric sphincter)
of rats. After six hours they were able to extract material corresponding
in UV spectrum to an imidazole, resultant from the reaction of an aldehyde
with the dye. Separately the authors administered a single dose of C-14
labeled dye to rats and examined the feces and urine for four days. Of the
total activity excreted, 87.5% appeared in the initial two days; of this
82.2% was in the feces and 17.8% in the urine. Chromatography of the feces
extracts revealed unchanged dye, and dye with a hydrory at the 6 position
of the naphthyl. In still another test the rats were given the dye orally
at the same time an i.m. dose of S-35 sulfate was given; both bile and
urine were collected. In the bile were found six colored and one colorless
metabolites: unidentified (S-35), unidentified (no S-35), l-(o-tolylazo)-
70
-------
6-hydroxy~2-naphthylamine-0-hydrogen sulfate N-glucuronide (S-35), l-(o-
tolylazo)-6-hydroxy—2-sulfamin.onaphthalene-O-glucuronide (S-35), l-(o-
tolylazo)-6-hydroxy-2-naphthylamlne-N-glucuronide (no S-35), l-(o-tolylazo)-
2-sulfaminonaphthalene (S-35), and a naphthalene derivative of reduced
azo (colorless, no S-35), In the urine were found three colored and three
colorless metabolites: unidentified (S-35), l-(o-toly!azo)-6-hydroxy-2-
sulfasninonaphthalene-0-glucuronide (S-35, also found in bile), l-(o-tolylazo)-
2-sulfaminonaphthalene (S-35, also found in bile), unidentified (colorless,
no S-35), unidentified (colorless, no S-35), and unidentified (colorless,
S-35).
Sato et al (1966) investigated the occurrence of N-hydroxy metabolites
in the urine of laboratory test animals given parenteral doses of carcino-
genic and non-carcinogenic members of the 4-aminoazob3nzene family. Their
findings are given in Table 17, The authors were able to synthesize N-
hydroxy-4-aminoazobenzene, but found it somewhat unstable even when kept at
0-5°C under a nitrogen atmosphere,
Ryan and Welling (1967) gave a single oral dose of pure Sudan HI, or
a single i.p, dose of pure Sudan III and Sudan IV to rats. There was no
excretion of either, or any metabolites, via the bile or urine. From the
oral dose of III, 84-951 was recovered in the feces, unchanged. From the
i.p. dose of III, 6% or less, depending on amount given, was recovered in
the feces, unchanged. From the i.p. dose of IV, less than 3.5% was re-
covered in the feces unchanged. When an i.p. dose of III tritiated in
the terminal benzene ring was given, after 96 hours orly 16% of the ac-
tivity had appeared in the urine (only 9% in a female), 5% in the feces
(21 female), and < 1/2% in the bile. The major metabolite (80%) in the
urine proved to be 4-aminophenol. The authors commented that the failure
-------
Reprinted with permission from Cancer
Research 26(8):1678-87 (1966). Copyright
by Cancer Research Inc., and the
American Association for Cancer Research.
TABLK 17.
THE UHINAHT EXCHKTIOX (IF A'-AcEr\-i.-4-AM!Nii.wur.E\zKNE ANI> lid A'-, 3-, AND 4'-ITiiiiiofY DKIHVAI i
BY RATR, MICE, A.VD HAMSTERS .U'IEK ADMINIKTIUTION or AMINOAEH })V».K-
MIIA
Bat
V..,!l,f;
lilllloler
Srx
V
M
M
M
M
M
M
M
M -
M
M
F
F
F
V
M
M
M
CoMPULND I.NJCCTLO AST! ROUTE6
AAH (H c.)
AAB (i p.)
AAH (i p )
A' !l\drn\vAAB (i.p.)
A' llydn.vy AAH (i.|>.)
All (i.|>.)
AH (i.p.)
MAB (i p.)
HAH li p )
DAH (i.p.)
A'-Ilvdroxy-AB (s.c.)
AAH (i p.)
A"-lhtfrov; AAB (i. p.)
AH (i p )
A'-lhdr.jxy-AB (s c.)
AAH (i p.)
Ar-H;,dro..v-AAB (i p.)
AB (i p ;
Uirr
Control
Control
);)
„ .__ _ ,
AMI
0 .11
0.18 :t O.O/*
o n
0.17
0.71
ii.U.I
n.07
o.ot
0.07
0.03
(I.O.'i
0 (i!)
(1 80
n.i.
n i.
0.01
0 15
o.in
% OF TH>Sfc F
.
A'-Jfydroiy-AAB
0.30
0.25 -i- 0 OB
0.22
0 !)2
0. 11
0.02
0.10
n.i.
O.Ofi
0.02
0.17
2.4
21.0
0.12
n.i.
2.3
b.'i
2.1
V *«TF.O »1
,ni>d,,u,.AA!l
O.(<0
0.3.) i t) 17
0.30
0 2!
0 ?H
(l.HS
0.17
0 02
0.13
0.0(5
0.15
0.1(1
0.12
li. i.
n i
0.13
0 IS
0.32
* I h" iililiti-viiit 1011:1 iHi«d IIH • AH, -1 :iiiiiiii).ufili..||/,rt)«!; MAI*>, A' nulhvl l-i'iniintiwdlxiimcnc; HAH, A",A'-l 4-nin!ii(..t/.ol)c-n/riif; nlm-du(., ril><«l!;ivin-di-Jicinit..
•liiji-i In,i, Hchc>.i ylin (("<" S i.i|;/ml), Mid O.M or C ?() ml w a* itijrrd-i} h.,- Infi- mi«
JUKI1, rc-pi'divcly. The other mule; rat.t \M rf inj''ctcd i.)). lit 0 and (i hr with (in itn .unit, of dye I'qtiiinolnr to 1(1 n:p; of \ \U/1'A' >
m'; 'Ai'i^ht; Sii 5 inn of A \H (.r an cqnivi]!"i.l, inno'uit of fi'iolhtr tly1 W.TC en-pi i ;(! per n.l of 0 'J'"t, nnd in in eld or id" Mniut io.t 'l
ft" ale ruis n'eoived 30 mn of AAB in 0 1 nil of liii,apryiin. Tin luiouwere ii>jn l(d OIK i: w itli 0 2 nil of u tneii]>rylin sus» n ">;i (< '('J1
ite.', to 1(1 nig of AAB/ml), while the Inn,-If \» leevived 0.21 i,.l of n trir.'iprylin suKprnsion which contained 50 mjt of AAB or
C^.i-.'lf-jit uitiounl of Al> or A'-h}diox\ -\/\B/ml
'Hit iiiiniheis in ])iiienth(.se<-. iKnoli1 (he Nf> of anirniils \,)u«<- urine writ1 pooled for 'hij anutyxrH.
Avcr.it'e d- the piolmbte c nor
*B i. fiiol identified) iii(.r,iulii s that no m<" Uiiioiitu rould bo iili iitified. The totiil almurplion in Ihenr- nreiis notioni'.y «iri'/n:i ted
fcuthai, OOl'/i of I he tlvc (ulinitiislered. Ill t!»% case of mici; fidministcred ritlior All or AT-hydiuxy-AIl, however, in unideiiiii'
m>'.lntiy IIIIVP iieco'inlcd lor i;!.mtt 0.1% of the. ndiniui»ler«:d uoinpoiind.
to find this metabolite in the urine of rats dosed with unlabeled dye
resulted from the very low quantity involved.
Fore et al (1967) found that Brown FK (a complex mixture) was only
decolorized (azo linkages reduced or broken) by the contents of rat caecum
or distal small intestine, and not by stomach or proximal small intestine
contents. Sulfanilic acid and a material similar to aminophenazines
(producable from polyaminobenzenes by condensation-oxidation) were
72
-------
recovered from these in vitro experiments. The accumulation of the break-
down products inhibited further breakdown of the components of the dye. In
Table 18 are the results of i.p. and intragastric dosing of rats with re-
gard to appearance in caecum, feces, and urine of unchanged components of
the dye (A and B bands), sulfanilic acid, phenazine-like material, etc.
Only traces of the phenazine-like material were actually excreted after
intragastric dosing, the ultimate fate after creation in the caecum being
unknown. Similar results derived from daily oral dosing of pigs for three
months with 100, 250, and 500 rag/kg of Brown FK; traces of the phenazine
material were found in the intestines at the two higher dosages. With
rabbits given 1-9 daily doses of 1 g/kg the phenazine material showed up
only in the urine, and in more than trace amounts; otherwise results were
similar to those from rats and pigs. With guinea pigs given 3-11 daily
doses of 1 g/kg, the phenazine material showed up in the caecum. Prior to
the experiment with pigs the authors would not have expected the finding
of sulfanilic acid and the phenazine-like material in rats to have had
much meaning relative to the toxicity of Brown FK in humans. However, with
this finding they considered it of importance to determine just which bac-
teria were responsible and how widespread, interspecially, they might be.
Hanaki (1967) reported that rats fed N-methyl-N-:'.sopropyl-4-amino-
azobenzene excreted 4-aminoazobenzene and N-isopropyl-4-aminoazobenzene in
the bile and urine. Also, by incubating the N,N-diallyl parent with rat
liver homogenate, the methyl group was the one to be cleaved. However, the
polar dye isolatable from the rat liver was shown to be N-methyl-4-amino-
azobenzene.
Walker (1970) had a good review of the literature, inclusive of ex-
cretion, into 1969.
73
-------
Table 18. Chromatograpine findings* with urine, faeces and caeca! contents^- of rats given multiple doses% of Brown FK
Specimen
Urine
Faeces
Caeca i
contents
Route
(mg/kg/day)...
Principal Interval after
finding? last dosefhr). .,
Bro\\.i FK (B bane')
*Bros\n colour*
Su'plurniic acid
'Blue material"
UV-fiuorcsceiit spots
Yellow snot
(ahead of A band)
Ero-.s:-. Tl< d\ band)
3!--n-.i fX tl< hand)
S.iq- M- lij ;;c J
'Cor.jiica'cJ colour'
'Polymeric material'
Pher.az'ne-I'ke material (P)
l'\ -D'—Tcsccrt ?r!o;s
!::o-s- --•:«:' (A Vind)
B"o\.n rl< (B band)
Sulpha;:!!;:: arc!
'Conjugjted colour'
"PoKmeric material'
Phena?ine-like material (P)
UV-fiucrescent spots
Intragastric
. 0-?
0
Tr—
+
0§
1
0
0-Tr
Tr — ~
Tr-4-
0
Tr-1
0
0
-•-
Tr-+
0-4-
Tr-1
100
0-6
0
Tr— i-
4-
°§
1
0
0
~r — u
Tr— f
0
Tr
0
C-Tr
-i--f
+ -f
0-Tr
1
18-24
Tr
4-
Tr
0
0
0
0
Tr
0
0-3
Tr
Tr-+
+ --H
0§
1-2
0
0-4-
_J j__ __ '
+-+H
Tr-+
1
0
+ -j-
4-+ +
-r + +
+ +
1
1000
0-6
Tr
-f
4.4. j.
Tr-+§
' 1-2
0
-!--f
r -f 4- -*- 4-
-+ J-4- +
Tr
1
Tr — t- + +
_; > i
+ + +
-(.-(.+
+ -!— + +
I
18-24
Tr
+ 4-4-
•f -f-t-
1-2
Tr
*T~
4- 4-
+ + +
+
Tr
2
0
0
+
+
T-
+ 0
0
100
18-24
Tr
Tr
0
Tr
0
0
0
0
0
0
Intraperitoncul
0-3
0-4- 4--
0-4-4-
0-4-
0
1
4-
0
0
Tr-4-
0-Tr
0
1
0
0
4-
0
0
2
500
0-6
"T — i — r
4.4.
4-
0
2
4-
0
0
Tr-4-
0
0
J
0
0
"T"
0
0
2
1000
18^24 18-24
0
0 4-4-
4- T-
0 4-
1
0 Tr
0 Tr
4- Tr
Tr +
0 0
0 0
0
0
0
4-
0
0
0
0
Level in diet (%)
(1-. 3-&12-vvk
results)
0-001
0-6
0
0
0
0
0
0
0
0
0
0
0
001
0-6
Tr
Tr
Tr
0
Tr
Tr
Tr
0
0
Tr
0
O'l
0-6
Tr
Tr
+
0
4-
4.,
Tr
Tr
Tr
Tr
0
1-0
0-6
+
4-
4.4.
4-
4.
Tr
4.
4-
4-
4.
0
*3ymbo!s v,sed: 0, no difference from controls \vithou',Brown FK; Tr, trace; +, +4- and + + -f present in small, moderate and large amounts, respectively.
tCacca! contents % c.-e obtained at autopsy, carried out at the end of the slated interval after the last dose of Brown FK,
JA to;a! of 10 daily doies was given by intragastric or intraperitoneal routes,
§Colour!css material giving yellow colour with Ehrlich reagent also present in this position.
Reprinted with permission from Food
Cosmet. Toxlcol. 5:459-73 (196777^
Copyright by Pergamon Press Ltd.
-------
Ryan and Welling (1970) dosed rats orally and parenterally, and man
orally, with the food dye Black PN. Qualitative findings of the excretory
routes of the dye and its metabolites are given in Table 19. The last
five columns on the right correspond to: azo metabolite, sulfanilic acid,
non-azo metabolite, non-azo metabolite, and non-azo metabolite, respectively
(all identified). There are two possible azo metabolites as there are two
azo linkages in the dye, but only one, apparently, was found. Residual
dye and the azo metabolite were found in the stomach wall and contents.
SA and DSA were found in the intestinal contents. None of the azo metab-
olite or dye was found in the bladder, intestines, heirt, liver, or stomach
after i.v. dosage. In Tables 20, 21, and 22 are the quantitative measure-
ments of excreted metabolites. It was not found possible to quantltize
Tabled Excretion of Black PN ami metabolites in rats and man dosed with the colouring
Route of Excretory
Species dosage route Black PN SNSA
SA
DSA ANSA AHNDA
Rat Oral Urine
Faeces
Inlraperitoneal Urine
Faeces
Oral Bile
Intravenous Bile
Man Oral Urine
+*
+
+
+
-r
+
4-
4- = Compound present
•Found only after single dose of 100 mg/rat.
Compound absent
Table 2ft Quantitative excretion of metabolites of Black PN after a single oral dose of the
colouring to rats and man
Excretion of metabolite (% of theoretical yield)
IMtctstsoI i tc
excreted
SA
ANSA
Excretory
source
Urine
Total
Faeces
Total
Urine/faeces
total, .
Urine
Faeces
Time
(tor)
24
48
72
43
72
24
48
20 mg/rat
36-7
6-9
0-9
44-5
29-5
5-7
35-2
79-7
with Black "N dose
100 ing/rat*
- 40-5
ir-8
2-5
58-3
30-2
2-8
33-0
91-8
-------
Table i'* Quantitative estimation of some metabolites in bile ami the gastro-intestinal
tract S lir after an oral dose of 25 nig Black PN/rat
Metabolite
SNSA
SNSA
Black PN
SA
Source
Bile
Stomach 4- contents
do.
Intestine + contents
Theoretical yield of
metabolite (%)
Trace
3-t
18-4
10-1
itcitive excretion of some metabolites of Bl.ick PN after intrjperitoneaf
injection zf the cu!u:ir;ng to ro/j
Metabolite
excreted
SA
Black PN
SNSA
ANSA
Excretory
source
Urine
Faeces
Urine/faeces total
Urine
Urine
Urine
Excretion of metabolite (% of theoretical yield)
with liiaek I'N do.ic (ing/raO of
T"im^ — . .
1 1 rue
(iir)
24
48
72
' Total
48
72
Total
24
24
24
10*
60-2
80
00
68-2
13-9
6-7
20-6
83-S
0-0
7-7
5-0
20"
:>4
5-9
o-o
65-3
20-1
4-0
24-1
89-4
0-0
8-2
2-0
100|
4j-7
15-7
3-5
65-9
19-0
5-9
249
908
06
60
*No lilack. PN in urine or faeces; no SN'SA in faeces.
fNo Black PN or SNSA in faeces.
Tables 19-22 reprinted with permission
Food Cosmet.Tpxicgl. 8:487-97 (1970).
Copyright by the Pergamon Press Ltd.
the metabolites DSA and AHNDA because of their chemical instability once
isolated. Production of sulfanilic acid apparently only occurred in the
intestine, as a result of bacterial action. The rat's enzymes did not seem
able to reduce the azo bond which would result in this particular metabo-
lite, but they did reduce the other azo link.
Gingell et al (1971) investigated the influence of intestinal bacteria
on the reductive cleavage of the azo bonds of Prontosil and Neoprontosil
by treating rats with antibiotics prior and subsequent to dosing with the
azo compounds. In Table 23 is the comparison between control and antibiotic
treated animals with regard to excretion in urine and feces, and nature of
the metabolites, showing the strong effect of the treatments. In Table
24 it is seen that, with Neoprontosil, the bacteria are involved even when
76
-------
the dye is introduced i.p. When biliary-cannulated rats were given an i.p.
dose of the S-35 Prontosil, 48% of the dose was excreted in the bile after
48 hours as the glucuronide, and only 3% as sulfanilamide; when the dose
was oral, these figures were 23.5 and 1.9%, respectixely. When Neoprontosil
was dosed orally to these altered rats, 1.4% of the glucuronide appeared
in the bile after 24 hours, and 14% appeared as sulfanilamide in the urine
at the same time; when the dose was i.p., these figures were 65 and 14%,
respectively; when the dose was i.v., these figures were 67 and 9%, respec-
tively.
3. transport
Ryan and Welling (1967) showed that unchanged dyp. could be recovered
in the feces of rats given i.p. doses of the dyes Sudan III and IV. Since
there was no biliary excretion of the unchanged dye, there must have been
diffusion through the peritoneum and intestinal wall.
Ozkan (1970) showed that 3'-methyl-4-dimethylaminoazobenzene could be
transported across the placenta in rats, producing changes in the liver of
the offspring in line with the amount of dye ingested by the parent.
Golub (1971) demonstrated that o- and p-aminoazouoluene could be
transferred across mouse placenta.
4. distribution
MacDonald et al (1953) fed rats N-methylated (C-14)-aminoazobenzenes
and measured the distribution of radioactivity in the g.i. tract and in
liver proteins - Table 24; in the table MAB and DAB are abbreviations for
4-methylaminoazobenzene and 4-dimethylaminoazobenzene.
77
-------
Table 23-The distribution of 3iS in rats receiving [3:S] Prontosil orally -with and
without treatment with antibiotics
The oral dose of ['"S] PioMto-.il hydrochloride wjs 56 mj/kg and each rnt received
10 /id of -1-'S. The antibiotic treated rats each received orally ncomycin sulphate (100 mg),
b.!ti;racin (50 1115) :ind tetrarvdinc Jndrnchloi ide (50 1115) tvvicc d.iily for two days be-fore
Piontoiil. The antibiotics were then given 4 h before and 4 and 24 h after the administra-
tion of Prontosil. The urin.uy metabolites were separated by paper chronntography and
radiochromatogram scans piep ired. The metabolites were determined by cutting out the
r.ppioprntc areas from the paper and counting the areas in the scintillation spectrometer.
The figures given are the a\nv,i;e values for three rats with ranges in parentheses.
% Dose of 35S found in
Days after
Material examined dosing
Urine
F.eces*
Liver + lung + kidneys 4- spleen
Gastointestinal tract + contents
Rest of carcass
Tet.il of above items
O" prwnfj of urine
Prontosil jY-fjIucuronide
Total sulphanilarnidef
Sum of above components
2
2
2
2
2
2
0-1
1-2
Total
0-1
1-2
Total
Total
Control rats
81(78-84)
2-1(1 -7-2-
1-8(0-4-2'
3-3(2-2-4'
4)
9)
3)
2-4(2-3-2-4)
88(84-92)
4-6(3-5-5
0-8(0-6-0-
5-4(4-3-6'
60(52-71)
14(8-21)
74(72-79)
79(76-85)
•6)
9)
'2)
Antibiotic-treated
rats
43(41-45)
4.4(5 .?_(
1-8(1-0-3
36(30-45)
3-2)
•4)
6-9(5-4-9-6)
92(90-96)
1(1-1)
. 3(2-3)
4(3-4)
6(5-5)
33(31-35)
39(35-41)
43(39-45)
Tables 23 & 24
reprinted with per-
mission from Xeno;-
biotica 1:14 .-56
(1971). Copyright
by Taylor & irancis
Ltd.
* At 'east 80% of the faecal activity was present as sulphanilamide (free + acetylated).
1 Free + acetylated: in the control rats about 82% and in the treated rats about 90% of
th- t'.tai bu!ph: !(>"', "f f(ff) «"« f'itrr«( in the uri'ie nftfr lp.
78
-------
DISTRIBUTION OF RADIOACTIVITY IH HATS FED CERTAIN
N-METJI¥Iy-C'<-LABELED DYT.S
DAB
.V-llwllul-
1MB
M VII
Duration of experiments, hrs.
Per cent total activity:
In respired CO2, 5 hrs.
* , 10 "
In stomach und smnti intes-
tine contents
In e«'nm and large inlcstin*
and fcce*
M
a
86
13
S
M
10
SB
40
8
8
K
10
i5
47
5
C
V
10
85
.);•;
3
2
F»
10
19
30
8
6
M
5
23
20
8
M
10
SO
40
8
«
M
S
27
Irf
4
M
10
1R
4 1
7
4
M
^
1!.
*7
I
\1
1(1
i"»
64
5
fi
Standard
\,\\'(T protein
activilyf of:
JOO 140 450
800
Liver srrinc (protein-bound) 1,000 1,4(10 8,400 J.SOO
• choline
Per cent activity:
Of M rine in /J-carbon
Of rholme in methyl (
ho mo
7.".0 S.tlOO
Hl-l
K'M
4.CUO 5,001) 7,400 fl.OUO 9,400 8,800 5,300 4,WO 4,000 S.Hrt. S/ctO
08 104 100 100
68 77 70 77
100
OS
76
(13
80
Bi
81
Hi
* Pooled u
f Bt»ndard *peet6c activity ^count«/Riin/ln(l) •• —
from two imrDftlutr rati; »« "Methods,*"
X nhvt
Reprinted with permission from Cancer
Research 13:292-97 (1953). CopyrightT>y
Cancer Research Inc., and the American
Association for Cancer Research.
Berenbom (1959) fed male rats for four weeks on £ diet containing
0.06% 4-dimethylaminoazobenzene (DAB), then for 4-5 dt-ys using N-15
labeled DAB. In separate experiments the labeled N was as follows:
C6H5-N* = N-CgHitNMea (DAB-1) » C6H5-N = NA-CeH^NMea (DAB-2) , and C6H5-N
= N~CgHi+N*Me2 (DAB-3) . The rats were sacrificed and the homogenized
livers centrifuged into nuclear, mitochondrial, and microsome-supernatant.
In a different series of otherwise identical feedings, the three fractions
were further broken apart by solvent extraction. Tables 25, 26, 27, and
28 present the findings on the distribution in the liver of metabolites
of DAB.
Baba (1961) gave a rat 15 mg of BAB (C-14 labelled in the non-amino
ring) via stomach tube on two occasions in a single day, followed by a rice
diet containing 0.06% of the C-14 DAB for 72 hours (elapsed time from the
-------
initial force feeding). The liver was sectioned, rinsed free of non-
protein bound DAB, and then autoradiographed. Very little activity
appeared around the bile ducts. Distribution of the activity was nearly
uniform across the peripheral, middle, and central zones, slightly lower
in the peripheral. Activity in the two lobes was the same. Slight activity
existed in Kupffer's cells.
Radomski (1961) gave stomach tube doses of Citrus Red No. 2 to male
and female rats. After 24 hours no unchanged dye could be found in kid-
ney, liver, muscle, or spleen tissue. When the dose was 5 mg, there was
no dye in the fat, but there was 4-10 (Jg/g after a 20 mg dose. Starting
with sixteen rats, killing four after one day and three on succeeding days,
Radomski gave daily tubal doses of 150 mg/kg (100 mg/kg in a seven day
experiment) of the dye. He found that dye content in the fat dropped in
a linear fashion at the higher dose - none left on the sixth day from
15 ug/g after the first day, in a logarithmic fashion at the lower dose -
none left on the seventh day from 13 yg/g after the first day. This study
was repeated on external D&C Red No. 14 with similar results. More of
the dye was initially incorporated into the fat than with No. 2. Females
showing 13-39 pg/g after dose 1 showed 0 yg/g after dose 7; males show-
ing 13-47 Mg/g after dose 1 showed 0 Mg/g after dose 10 (6.5-24 Mg/g after
dose 7). Drop off was smooth in the females, zig-zag in the males.
Storey (1968) studied the distribution and retention in connective
tissues and bones of chlorazol fast pink and related dis- and trisazo
dyes (i.p. dosage). Two consecutive daily doses of the fast pink at 25
mg/kg to rats resulted in noticeable external coloration still obvious
after six months. There was no staining of 17-18 day fetuses. Noticeable
color in the urine persisted six months. Internally after one day staining
80
-------
was obvious in skin, fascia, muscle attachments, most internal organs,
aortas, and bone marrow. Also colored was the cartilage in the nose, ear,
and trachea. No staining of the brain, spinal cord, eyes, nails, or hair
occurred. At six months coloring had become faint except for the aortas,
which were still bright. Young, but not old animals showed staining at
the margins of cranial sutures, dentine, and long bone growing metaphyses.
Gingell et al (1971) examined the distribution in the bodies of rats
after oral dosing with Prontosil S-35. The results have already been dis-
played in Table 23 in connection with excretion of metabolites. Treatment
with antibiotics to study the effect on in-gut breakdown did not change the
accumulation in the internal organs, but it did increase the amount in the
carcass by a factor of 2.5-4.
TABEK 25.
QL'A VflTATIVK IllTi.KKXCK DATA TOR RATS L'SKI) IN N15-I,AHKI.KL> DAH K\PK.RI.MEXTS
SK>IIV.<*
I* _
W
DAB
1
2
3
1
2
3
Av.
ui.ny wr.
1.55
10.5
125
100
122
1S6
Av.
Ll-i Ml WT.
8 8
:> 4
7 1
4.8
5.9
7.1
TOTAL X
(MKQ/&W
•WKT WT.J •
1 ,'3!>
1 21
1 07
I. SO
l.!H
1 K TIIK
J.IVKll OK HATS Kni) X15-I.AilK.I,KI) DAH
M'KNlWNT
(*M,I,.H ])
DAIM
DAH-2
DAH-3
t't'.tt r>.s
,
X
1 t
1 0
2.1
T KM KM
M
1 0
:( 5
2 9
i X Id1
_
S
2 n
•t i
3.7
N
8
10
8
N1* IN MVKH
M
22
27
24
— .
S
70
G.1
OS
Kiirli viiluc is (lie average of tliroe vvporlineiil.s.
N = nuclei, M = inilocliondria, S = microsoine-Mipernnlnnl.
The N" ciiiicenlralion lias liccn corrected for Uic nntural
rontent of N" of the pnrtirulnr fractions nsdelcrminedoBral'
fed the diet containing nonisolopic DAH.
81
-------
TAHLK 27.
RI:I,ATIVK COXTKNTRATION OF X1' i\ TIII: Xrnr.ui
MiTOCHONDIUAL, AM) MlCilOSOMK-StTEKVATAST
FKUTIONS OK THK LIVKK OK H\TS FED N'5 <,ABI:I,KI>
DAI)
flEHIF.S 1)
DAB-1
DA15-2
DAU-3
N M S
10 1.4 i 1
10 1.8 22
10 14 1"8
.^KWHAT.0*
N .\t
1 0 1.0
14 IS
15 15
S
1 1
1 t
i :
N — nuclei, M---mitochondria, S = iiiicrosome-siipprnat(iiit.
TAHI.E 26.
DlSTIlIllliTIOX OK XlTllOGKN AM) N" IN CHBMICAI. CONSTITUKN'IS Ol'
NUCLEAR, MITOCHONDIKAI., AND SUPERNATANT KJIACTIONS OK
THK LlVKH OF HATS FKD N"-LAItELKO DA1J (SKRIKS II)
MATHIIAI.
Nuclei:
Cold acid-soluble
l.ipitlc
Nucleic acid
I'rolcin
Ciild arid-Kdliillln
l.ipide
Nwlt'ic »cid
I'rolein
Micro.somc-.supcr-
n.'it.'inl:
Cold acid-solu)>lc
l.ipide
Nuutcif ncid
IVtrfh
TOTAL NI
WBT WT
DA 11-1
O.H
9.7
(i.V
11. 4
1.11
11.11
4 .7
«8.-t
10.4
IW.7
fl.ll
«7 . :t '
TIKHJfN, MKQ/(;M.
. Of' MVKHXIO1
DA IK
0.0
8.0
7.4
10.4
1.4
1 1.7
1.4
SJ.3
10.4
.'11.7
(l.ll
/57:i
DA H-.l
0.8
10.3
(!.7
7.0
1.4
11). H
:i.-i
ill 4
H.H
:t.r> . o
7.4
«4.'H
A fOM PKH CKNT
KXCK8H N'»X10J
DAII-1 I)AH-« DAI
:i.l
a o
0*
1.0
4.7
si.ll
'i .H
1.0
4.4
!2.H
11. 'i
4 t>
4.0
5 H
0*
4.0
4.C
M.4
11 .7
4.n
.i.:*
:l . 0
!).H
r.«
4
4.
()'
:t
4.
:i.
:i.
2
V.
:i.
4.
4:
l.ll
Ml
* Too low to measure nccunitcly.
Tlic Nlf oinccnlriiliun has been corrected for (lie milurnl cant nit of N" of the
piirlicuhir fractions ns determined on ruts fed the diet containing nonisotopic
Tables 25-28 reprinted with permission
from Cancer Research 19:1045-49 (1959).
Copyright by Cancer Research Inc., and
the American Association for Cancer
Research.
82
-------
B. Physiological effects
Danneberg and Schmahl (1952) tested the estrus-inhibiting properties
in rats of a number of azo compounds. The compounds which had this property
were not necessarily also carcinogenic. Strong estrus inhibitors were:
4-aminoazobenzene (AB), N,N-dimethyl AB, N-acetyl AB, 4,N,N-trimethyl AB,
2',3-dimethyl AB. Non estrus inhibitors were: 4-methoxy-N,N-dimethyl AB,
4-nitro-N,N-dimethyl AB, 4-(phenylazo)-N,N-dimethyl AB, l-(4-dimethylamino-
phenylazo)-2-naphthylamine, l-phenylazo-2-naphthylamine, l-(2-methylphenylazo)-
2-naphthylam±ne, l-(2-methoxyphenylazo)-2-naphthol, and 2,4-dihydroxyazo-
benzene (4,4'-dihydroxyazobenzene is an estrogen).
Lacassagne et al (1952) fed adult rats four derivatives of azobenzene
as 0.06% of their diet until death occurred. All four caused considerable
weight loss. In order of decreasing damage to the liver, the compounds
were: 3',N,N-trimethy1-4-aminoazobenzene, 4'-N,N-trimethy1-4-aminoazobenzene,
4'-phenyl-N,N-dimethyl-4-aminoazobenzene, and 4-hydroxyazobenzene.
Adams and Roe (1953) applied to the skin or injected beneath it solu-
tions of azo compounds to study the effect on hepatic catalase activity.
None of the compounds caused any damage to the liver itself. In decreasing
order of ability to depress the enzyme level the compounds tested were:
3',N,N-trimethyl-4-aminoazobenzene, 4-N,N-dimethylaminoazobenzene, 2',3'-
dimethyl-4-aminoazobenzene, m-azotoluene, 2-amino-5-azotoluene, and azo-
benzene (no depression). Doses applied were 10-15 ym.
Takahashi (1953) reported the following azobenzene compounds to be
estrogenic in mice (compound, dose in Jig, % of mice responding): 2,2',U,UI-
tetrahydrcoy, 20, 100 (10, 60); U,li'-dlhydroxy, 1000, 80 (500, 20); 2,U-dir
hydroxy, $00, kO', 2,li-d±nydrojy-2',5l-dimewic«y, 1000, 0.
Reiss et al (1954) gave 80 mg of 3',N,N-trimethyl-4-aminoazobenzene
-------
over a four day period to male and female rats in a vitamin poor diet. In
both sexes the liver showed higher BI and lower 62 levels than controls.
A similar result obtained with vitamin C, but the females were dependent
to some extent on the vitamin content of the pre-experiment diet.
Akai and Yasumori (1955) cultured the fungus Cochliobolus miyabeanus
in a nutrient solution containing 10 yM-1 mM Congo Red or 0.1-0.5 mM Chrys-
oidin. Maximal growth occurred at 0.25 mM Congo Red (39% higher than the
control) and at 0.25 mM Chrysoidin (107% higher than the control). At the
highest concentrations of both, growth was less than that of the control,
the Chrysoidin being the more toxic. Optimal usage of glucose and nitrate
occurred at the lowest dye concentration, not at that concentration which
gave highest growth. Innoculation of the dye-grown fungus on rice plant
leaves showed that there was decreased toxicity to the plant; the decrease
was independent of the Chrysoidin concentration, but correlated with the
Congo Red concentration (the lower the concentration the higher the
toxicity).
Nomura (1955) showed that 2',4,4'-trihydroxy-2-methylazobenzene
showed 100% estrogenic activity as a 50 yg s.c. dose in castrated
mice, 0% as a 30 \ig dose. The same figures applied fcr 2,2 '-dimethyl-
4,4'-dihydroxyazobenzene. In the case of 2 ,4,4'-trihydroxya;jobenzene or
2-methyl-4,4'-dihydroxyazobenzene, a 300 yg dose had no effect.
Doi (1957) fed rats 0.06% of 4-N,N-dimethylaminoazobenzene in their
diet for 30 days. Examination of the liver showed increases over normal
in haemosiderin, ferritin, and ascorbic acid, decreases in catalase,
copper, and riboflavin.
Okuda (1959) fed rats a diet containing the usual amount (probably
0.06%) of 4-amino- or 4,N,N-dimethylamlnoazobenzene over a month's time
-------
and measured urinary excretion of B vitamins throughout. No reliable
change in thiamine resulted. Nicotinic acid increased slightly. Pyridoxic
acid decreased noticeably. Riboflavin increased considerably. Guinea pigs
did not show the increase in riboflavin.
Neish (1959) gave single i.p. doses of various azobenzenes to female
rats, in molar amounts corresponding to 165 rag/kg of 3',N,N-trimethyl-4-
aminoazobenzene. Methaemoglobin from tail blood was then determined at
3, 7, 22, and 28 hours. Table 29 sums these four values and presents them
as methaemoglobinemia. It may be seen that there was no correlation with
carcinogenicity.
Table 29. Methaemoglobinemic Activity of Some Azo Compounds
Azo Dye
A?o
4 -a
4-n
4-n
4-n
2'-n
not'iyinminoa/obcn
uomethylaimnoaz
!iocth>lammoaxob
rthylcthyl.iiiiinoaz i
ithvl-4-tIinn th> la
•th\l-4-Onnrtli\la
-4 '-<.' lhvl-4-diiuotiivl.ini
> Mi/onc ....
rnzcno ....
loazobcnrcnc . .
loa/obcn/r-ne . .
o.j/obeit/cnc . .
Carcino-
gcnicity
±
±
f + +
Mcthaemo-
globmemia
0
140
113
123
126
1SO
38
53
0
0
Reprinted with permission from
vnssenschatten 46:535 (1959)
by Springer Verlag.
Natur-
Copy right
Mascitelli-Coriandoli (1960) fed rats a diet containing 0.064% 3',N,N-
trimethyl-4-aminoazobenzene. After three weeks and six weeks, respectively,
the hepatic riboflavin had fallen by 1/3 and 1/2. Corresponding figures
for hepatic azoreductase activity reduction were 1/4 ana 1/2.
Yamada (1960) gave rats i.p. injections of Trypan Blue once a day
for three or six days (A or C), and twice a day for three or six days (B
or D). Radio-iodine was given s.c. six hours before autopsy. Thyroidal
uptake of the radio-iodine was 55-60% of the control in A, B, and C, but
only 14% in D. Thyroid weight ranged 70-80% of normal, D being lowest.
Pituitary weight of D was 90% of control. Adrenal weight was 135-155% of
-------
the control, increasing in the order A, B, C, D. Testis weight was
of control. Total iodine and protein-bound iodine in the serum were 62 '
(25.0) and 45.8 (25.0)% in C (D), respectively. On average, a single in
jection of trypan blue inhibited thyroid hormone secretion for 14 hours.
Boyland and Grover (1961) measured urinary ascorbic acid excretion
after 100 mg/kg doses of some azo dyes. The greatest percentage increase in
excretion resulted from 4',N,N-trimethyl-4-aminoazobenzene, followed by
the 2,N,N-, the 3',N,N-, and N,N- itself.
Neish and Rylett (1963) gave rats i.p. injections, 16.5 mg/100 g, of
azo dyes, and measured the hepatic glutathione level 24 hours later. The
glutathione (dye)/(control) ratios found were : 1.97 for _3_' ,N ,N-trimethyi-
4-aminoazobenze'he, 1.21 for the 4.' isomer, and 0.41 for the _2 isomer.
Neish and Rylett (1963, pp. 1147-50), in a follow up report on the
effect of 3',N,N-trimethyl-4-aminoazobenzene on rat hepatic: glutathione,
reported that 24 hours after an i.p. dose of 16.5 mg/100 g the stomach
was noticeably dilated and filled with food.
Kizer and Howell (1963) reported a study on the effect of 3'- and 4'-
methyl butter yellow on rat hepatic kynurenine hydroxylase activity. Ax-
though the control diet seemed to be deficient in something which also
affected the enzyme in the same direction as the azo compounds, the
latter's effect was still noticeable. The results are in Table 30.
TADLB 30* Effect of a low and a high carcinogenic derivative of d-dimethviaminoazobenzeno on the kymtrenino
hydroxylaso activity of rut liver-
En/ymo
Diets
Bnnal diet
BiiMiil diet with
0.0(1% 4'Me-OAn
0.0(1% 3'Mo-DAli
0 Weeks 4
(3) f 0. 51 ± 0. 04$ (3) 0.
(3) .
(3) .
activity*
Weeks
32
14
14
±
±
±
0.05
.04
.07
(3)
(3)
(3)
8 Weeks
0.46
. 27
.0!)
± 0.06
± .04
± .03
(3)
(3)
(3)
12 Wfcks
0.51
. 31
. 10
± 0. 0!)
-J- . 10
± . 11
'nMolc.i ]-li>
-------
Kline and Clayton (1964) fed rats 0.064% of their diet as 3',N,N-
trimethyl-4-aminoazobenzene, and measured the hepatic lactic dehydrogenase
activity. The reduction in activity did not become significant until the
livers had shown signs of cirrhosis for three weeks. Continuing the feeding
until tumors appeared in the liver revealed that the tumors had far less
activity than surrounding tissue.
Furlong and Thomann (1964) fed rats 0.06% of their diet as 3',N,N-
trimethyl-4-aminoazobenzene and measured the hepatic DNA polymerase. After
six days the activity had nearly doubled. By 20 days the activity had
peaked at about 2 1/4 times. After seven weeks the activity was only
slightly higher than normal.
Dijkstra (1964) gave rats a single intragastric dose of 2,N,N- or
3',N,N-trimethyl-4-aminoazobenzene and measured the hepatic ascorbic acid
level. The level remained in the normal range for the initial 40 hours
after dosing with the 3', then fell to only 60% of the mean. Between 10
and 30 hours after dosing with the 2 isomer, the level was above the nor-
mal range (about 10% higher than the mean); after the 35th hour the level
was the same as with the 3' dosage. The levels were still below normal
after two weeks.
Dijkstra and Pepler (1964) fed rats diets containing 4-aminoazobenzene
(AB), N,N-dimethyl AB, 2,N,N-trimethyl AB, and 3',N,N-trimethyl AB in
amounts equivalent to 0.06% of the N,N-dimethyl AB. Over an entire 20
week period hepatic ascorbic acid was above the normal range with the
3'N,N compound, especially from the third week. With che 2,N,N compound
the level was at the normal upper limit or slightly above. With the N,N
compound problems of interpretation of trend arose because of very wide
ranges from the 7th through 14th weeks, but from the fourth week the
87
-------
level averaged at or above the upper normal. With >B a normal pattern
was seen.
Manchon and Lowy (1964) demonstrated that rats fed a diet deficient
in vitamin B2 survived longer and showed better growth when a 2% solution
of Sun Yellow in water was their source of water. Omission of all B2 from
the diet resulted in death however.
Mel'nikova and Selikhova (1965) found that mice given azobisformamide
produced less serum pseudocholinesterase and acetylcholinesterase, and
less hepatic cholinesterase.
Mulay (1966) fed normal, three month old rats a low-protein, low-
riboflavin diet containing 0.06% 3',N,N-trimethyl-4-aminoazobenzene for
2, 5, 7, and 17 weeks.
TABLE)).Effect of ITopntOfnrcinopenic Diet on Aclronnl fllnnd tind Li^cr Chemistry of M.ilo
Oaborne-Meiidcl RnU. Each 7.alup is a mean of ilotcrmiimtions 011 10 rnts. Standard error fi,r
each value is within 2% of rvspeftire mean.
Time,
Diet wk
Purina chow (control)
Hepatocarcinogenie 2
5
7
17
Semi-aynthctie 6
Ascorbic
acid, mfj/g
2.38
2.51
3.13
3.38
2.17
2.98
Steroui.H,
mK/g
29.3
35.2
50.5
61.6
52.0
37.9
Fat,
186
193
257
280
290
194
Weight,
nig/100 g
body wt
11.7
12.2
12.6
12.8
15.5
13.7
» •"••! -\
Steroids,
niff/g
3.23
3.11
3.03
2.97
3.85
4.15
Vat,
mg/g
49.3
62.4
70.1
kH.fi
55.7
93.7
The results in Table 31 indicate higher steroid and fat content in the
adrenals onsetting at 2-5 weeks. The adrenal/body weight ratio was
noticeably lower than the deficient diet control through seven weeks.
Hepatic steroids and fat were considerably lower than this control. The
author claimed that hepatic ascorbic acid and ratio to whole body were
the same as controls, but did not specify which controls.
Sydow and Sydow (1967) gave rats a single oralrdose of 40 mg' of 3',N,N-
trimethyl-4-aminoazobenzene and then measured various hepatic functions
-------
over a two month period. There was no change in relative liver size, but
hepatic protein dropped about 20% during the first week. The protein level
was fully recovered within one month. Hepatic hexokinase was unchanged.
The glycogen content dropped by 2/3 in the first day, then gradually re-
covered, seemingly to a higher than normal level after two months (the
authors gave no P values). Glucokinase dropped 2/3 by the fourth day
(possibly even lower thereafter) but had recovered after one month.
Glucose-6-phosphate dehydrogenase dropped 1/2 by the fourth day, and was
nearly, if not actually totally, recovered by one mon^h. Arginase was
unchanged.
In a separate experiment the rats were given 5 mg of the azo compound
daily for 80-130 days. Relative liver weight was about doubled for 80 and
100 day treatments, but pentupled after 130 days. Hepatic protein dropped
20-25%. Glycogen dropped by 1/3 in the 80 and 100 day treatments, over
2/3's in the 130 day treatment. Hexokinase increased 2-2 1/2 times, but
showed a much wider range than the controls. Glucokinase dropped 76% after
80 days, 69% after 100 days (both having relatively high spreads), and 81%
after 130 days. Glucose-6-phosphate dehydrogenase dropped by 1/2 regard-
less of length of treatment. Arginase was unchanged. All of the changes
from 80 day treatment were at least partially reversiMe as seen from 6-8
weeks on a normal diet. After six weeks normal diet after 130 days on
treatment relative liver weight was returning to normi.l, as were the
glucokinase and glucose-6-phosphate dehydrogenase activities; on the other
hand, hepatic protein, glycogen, and hexokinase showed no change.
Doctor et al (1967) showed that rats fed 0.06% 3',N,N-triroethyl-4-
aminoazobenzene showed changes in hepatic and serum vitamin B-12 comparable
to those in fasted rats over the initial ten days. Over a 15 week test
89
-------
period the hepatic level gradually decreased in comparison with controls.
Grasso et al (1968) gave rats either a daily dose of 1 g/kg (by
stomach tube) for seven days, or a diet containing 2% for 12 weeks of the
British azo food dye Brown FK. Both treatments resulted in myofibrillo-
lysis and lysosomal damage followed by lipofuscin deposition. The lysis
is rapid and extensive after the forced feeding, occasionally not appearing
after dietary treatment.
Gaunt et al (1968) fed rats Brown FK at 0.001-1.0% of their diet for
21 weeks, and gave miniature pigs daily doses corresponding to 100-500 mg/ke
for 24 weeks. Unchanged were growth rate and food consumption, kidney and
liver weights and function. Hematology was normal. Apart from the follow-
ing, histopathology was normal. In the pigs the principal change was
deposition of lipofuscin in the liver in both sexes at all dosage levels,
accompanied by higher lysosomal enzyme activity. The lipofuscin also de-
posited in male, but not female, hearts. In the rats the 1% dietary level
caused this deposition in the heart, kidney tubules, liepatic Kupffer and
parenchymal cells, and skeletal muscle, being noticeable at 13 weeks in
females and at the end of treatment in the males.
Gaunt et al (1969) gave pigs 90 daily doses of Ponceau 4R of 100-900
mg/kg. The only effect noted was a slight decrease in erythrocytes in
males after six weeks at the highest dose.
Gaunt et al (1969, pp. 557-563) gave pigs 90 daily doses of Black
PN of 100-900 mg/kg. The only effect noted was development of mucus and
fibrin-containing cysts in the mucosa of the ileum of 1/6 of the 300 and
4/6 of the 900 mg/kg animals.
Beaudoin gave i.p. injections to 8-day pregnant rats of 14 mg/100 g
trypan blue, Evans blue, and Niagara blue 4B, and of 20 mg/100 g Niagara
-------
TABLE 3Z,
Total protein and protein-fraction concentration in serum of control and disazo dye-treated rats expressed as -mean values
in g per 100 tnl wiih standard deviation
Treatment »>
Control
8 10
10
20
Trypan blae l
8 10
10
20
Evans blue *
- a a
10
20
Niagara blue 4B '
8 9
10
20
Niagara sky
blue 6B *
8 8
10
20
Congo red *
8 8
10
20
Niagara blue 2B *
S S
10
20
Total
protcia
6.30*0.43
6,25 * 0.54
6.40*0.52
' 6.40*0,31
5.58*0.26*
6.73 ± 0.36 »
6.22*0,16
5.83*0.40*
6.20*0,43
6.55*0.26
4.90*0.33 *
7,25*0.34 *
6.12*0.43
5.80*0.41
6.52=: 0.36 *
. 6.05*0.29
'5,70*0.38 .
C.27*0.80
6.17*0.44
5.70*0.34
5.55*0.17
Gaisraa
, 1.09*0.33
" 0.87*0.28
0.49
0.74
0.5S
±0.14 *
±0.10
±0.10 *
0.58*0.14
0.61
0.64
0,38
0.73
0.63
0.65
0.90
0.80
0.88
0.72
0.53
0.34
0.75
0.65
0.38
*0.10
±0,12
±0,04 *
* 0.08 "'
±0.08
±0.14
±0.15
±0.16
±0.3S
i
±0.14
±0,33
* 0.04 *
±0.14
±0.12
±0.05 *
Beta
1.09*0.10
1.08*0.12
0.93*0.13 *
0.90 ±0.12 ,-
1.05*0.14 *
1.01*0.15
0.88*0,10
1.29=: 0.09 *
0.92*0.16 *
G.9S±Q.K
0.85*0.17
1.14*0.17 *
1.05*0.18
1.15*0.1?.
1.31 = 0.11 *
0.97*0.03
1.07 = 0.13
0.3u*U,i4
O.SB=;C.14
0.88*0.14
0.91±0.2S
Globulins
AlpUa-3
0.32 ±0.04
0.32*0.04
•t. 36 = 0.06
0,40*0.05
C.41*0 07
0.34*0.04 *
0.32*0.03
0.50*0.10 *
0,38*0.13 »
0,34*0,07
0,32=0.10
0.35 = 0.05
0.40*0 07
J 52 * sj Oo *
0,40 = 0.13
0.32 ±0.03
0.34*0.03
0.34 = 0.01
0.27 = 004
0.26*0.04
0,33 ± 0.08
Alpfca-2
0,35*0.02
0.30±O.OG
0.40*0.10
0.23 = 0X6
0.43 * C.07 «
0.25*0.05*
0.25*0.06
0.67*0.04 *
0.34*0.04 *
0.30 = 0.07
0.32*0.05
0.42*0.10
0.23*0.04
0."3*O.OS *
0.43*C.CO *
0.22*0.02
0.40*0.03 "
0.39 = c.;o
0.30=0,07
0.45 = 0.03 *
0.46=0,22
Aiph
0.78 ±
0,7G±
1.54 ±
0.85*
C.00 =
1.04 =
0.78±
C.S4 *
1.32 =
C.30 =
0,55 *
1.3 C-
0.33-.
oc^-..
j,2/,-i
0 72 r:
0.84 ~:
J..UJ. ~^~
Oi.2 '-
O.S-1*
1.46:--
0.
0
o.
0
0,
p (
0
0.
0,
^
c.
c,
0
0.
c
0,
0,
c
0.
-1
,02
.14
,22 *
.13
, JLU
,17*
.09
*'"'#
14 v
.09
.03 '
.J'i *
:2
v«
,."' ~*
.13
0°
.Ifa *
17
04
.12 '
Albumin
2.65
2.86
2,65
3.23
•2 20
3.48
3.3S
1.79
2.8Q
3.40
1.33
S.33
2.51
" C'.
2 20
3.10
2,50
2.63
3, IS
^ 52
201
= 0.38
2:0.36
±0.30
* 0.20
= 0.13 *
±0.3-1 *
±0.24
±0,30 *
±0,31 *
= 0.'lb
±0.10"
±c.t;c *
-• C o •
*r C ^
= '.) ~ ' '
*o-:j
± u.:-j.i "
= o.:-j
±O.C- i
= C.kG l
* C.w 1 '
»14 mg of dye per 100 g maternal body weight,
» 20 mg of dye per 100 g maternal body weight,
» Represents a significant change from the preceding value (P:
:0.02 or less).
Reprinted with permission from Teratology
2:85-89 (1969). Copyright by WTstar
Institute Press.
-------
sky blue 6B, Congo red, and Niagara blue 2B. Blood samples were taken just
before Injection, 48 hours after and 12 days after injection. Table 32
presents the findings on the protein content of the serum. In Section X.
B.4. the teratogenic effects found in this experiment are presented, but
the author did not think there was any connection between these and protein
metabolism.
Reuber (1969) gave rats five weekly s.c. injections of trypan blue.
Those animals which developed thyroiditis also weighed less than controls
or normal-thyroid experimentals; a greater percentage of females developed
thyroiditis, but those which did were closer in weight to "normals" than
the corresponding males.
Poirier and Pitot (1969) fed rats 0.054% of 2,N,N- or 3',N,N-trimethyi-
4-aminoazobenzene for up to 5 weeks. While controls showed a 48% increase
in weight, the experimentals gained no weight. Liver weights of controls
Increased 39%, of 3',N,N fed 24%, and of 2,N,N fed 78%. The 3',N,N ani-
mals after 2-3 weeks showed a loss of ability to produce ornithine-6-
transaminase and histidase (after casein hydrolyzate force feeding), and
also serine dehydratase. The 2,N,N animals after 3-5 weeks showed a loss
of ability to produce tyrosine-a-ketoglutarate-trnnsaminase and serine
dehydratase.
Decloitre and Meunier (1970) fed 0.06% of 4-d^.methylaminoazobenzene
for up to 12 months to male hamsters or the males of two strains of mice,
1C and C3H. The hamsters showed no hepatic cellular alterations. The C3H
mice showed glycogen and fat deposits in the hepatic cells after six weeks;
cellular damage became severe after three months, and continued to increase.
The 1C mice showed no hepatic alterations until five months. The glycogen
and fat deposits appeared at six months along with inflammation.
-------
Hepatic protein-bound dye increased slowly in hamsters to a maximum
at six weeks, remained steady for an additional six weeks, then slowly
decreased. In the 1C mice the maximum (slightly greater than in the ham-
sters) was reached in two weeks, then gradually decreased (lower than the
hamster level at six weeks). The C3H mice reached a maximum at three weeks,
about 2 1/2 times greater than the 1C mice. This decreased at a rapid
linear pace until three months, when it was between the IC's and hamsters.
Then it rose slightly at four months, and then dropped quickly to zero at
five months (at which point the IC's were slightly above zero, the hamsters
considerably above).
Over a five month period the hepatic azoreductase and NADPH-cytochrome
c reductase activities were measured. The hamsters showed a fluctuating
decrease of 10-42% in azoreductase over this period, and a decrease in the
other enzyme over the initial six weeks of 8-30%, and from nine weeks on of
40-50% (both non-time related fluctuations). The C3H mice showed an in-
crease in azoreductase of 10-40% from weeks 2-6, and a decrease of 40%
from months 3-5. Their C reductase increased 70-80% in weeks 1-2, then
decreased to a value fluctuating around normal from six weeks on. The 1C
mice showed an increase in azoreductase of 60% after one week, followed by
a quick return to normal; the level in months 3-5 seemed a bit below nor-
mal, but significance wasn't high. Their c reductase was 50% high at week
2, 30% high at week 3, normal from weeks 4-16, then 50% high at month 5.
Although C3H mice were known to spontaneously develop hepatomas after
one year on the control diet used, none of the control mice showed the,
apparently, irreversible cell changes occurring in the livers of the dye-
fed mice at six months.
Endo et al (1970) fed rats 0.06% of 2,N,N- or 3',N,N-trimethyl-4-
-------
aminoazobenzene. The former did not increase the level of hepatic muscle
type aldolase, but the latter did, even after only 15 days of feeding. If
the diet were maintained for 60 days, then the increased level of activity
was maintained for an additional 300 days.
Poirier and Pitot (1970) fed rats 0.054% of 2,N,K- or 3',N,N-
trimethyl-4-aminoazobenzene for up to five weeks in a low-protein, low-
riboflavin diet. The animals were then fasted three days, fed a 30% pro-
tein diet for 27 hours, and fasted another three days. The livers were
then examined for various induced enzyme activities. The 3',1I,N dye
resulted in zero or diminished responses of glucokinase, glucose-6-
phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme,
and citrate cleavage. The 2,N,N dye lowered the 6-phosphogluconate de-
hydrogenase and glucose-6-phosphate dehydrogenase, without affecting the
others.
Gaunt et al (1971) fed male and female immature rats 50-.5000 ppm
Orange G (British food grade) for 15 weeks. No effects on food or water
consumption or weight gain were seen. Heinz bodies were found in about
10% of the erythrocytes of all animals after two weeks at the 5000 ppm
level, and 0.8% after 15 weeks at 500 ppm. Hemoglobin was low at the
5000 ppm level after two weeks in the females, in both sexes after six
weeks. Methemoglobin was considerably higher (as percentage of hemoglobin)
in both sexes after two weeks at 5000 ppm. Packed cell volume showed a
significant decrease (< 10%) in both sexes after six w^eks at 5000 ppm.
Red blood cells decreased in females at two weeks, males at six weeks at
5000 ppm. Reticulocytes (as percentage of red blood cells) showed a large
increase in both sexes after two weeks at 5000 ppm. The serum was analyzed
for glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, lactic
-------
dehydrogenase, glucos .ire a nitroge-i, coial :>rotein , and albumin. The
only significant change at t.t, p<0.0i> Jfvel as a decrease in glucose in
both sexes at six weeks at the 5000 ppm level (but not at 15 weeks). All
internal organs were examined but only the spleen showed a weight change -
an increase (P<0.001) in both sexes after two weeks at 5000 ppm, except
that the heart, liver, and adrenals of 15 week females at 5000 ppm showed
increases significant at the P<0.05 level. On a "relative" base similar
results were found except that the significance increased to P "O.Ol for the
heart and liver, decreased below P<0.05 for the adrenals, and increased to
P<0.05 for the female gonads, and lieurn (P<0.01 at the 500 ppm level) at
15 weeks. In addition the adrenals of both sexes at 5000 ppm after two
weeks (only) showed P'0.05 increases.
The increase in spleen size was attributed to removal and break-down
of the Heinz-bodied erythrocytes. The reticulocytosis was attributed to a
compensation for the anemia resultant from the damaged erythrocytes. No
explanation for the increased adrenal weight, and only a partial one for
the lowered glucose level was offered. The authors concluded that the
no-effect level was probably closer to 500 ppm (25 mg/kg body weight/day)
than 50 ppm.
Gaunt et al (1971) fed Orange RN (British food grade) - approximately
a 6/1 dye mixture - to male and female immature rats at 60-6000 ppm for 15
weeks. Body weight and food consumption were unaffected. Both sexes con-
sumed more water at the 6000 ppm level immediately. Erythrocytes with
Heinz bodies appeared at two weeks in both sexes at the 6000 ppm level,
in both at the 1200 ppm level at six weeks, and in botb at the 600 ppm
level at 15 weeks. Hemoglobin dropped in two week females at 6000 ppm, in
males at 1200 ppm after six weekc, and at 600 ppm in females at 15 weeks.
-------
Methemoglobin (as percentage of hemoglobin) increased in both sexes at
6000 ppm after two weeks; lower levels were innocuous throughout the ex-
periment. Packed cell volume decreased at 1200 ppm in females after two
weeks, and at 6000 ppm in males after six weeks. Red blood cells decreased
in both sexes at 6000 ppm after two weeks, in males at 1200 pp>m after six
weeks. Reticulocytes (as percentage of red blood cells) increased con-
siderably at 6000 ppm in both sexes after two weeks, in both at 1200 ppm
after six weeks. Total leukocytes decreased at 1200 ppm (not 6000 ppr) in
females after two weeks, at 6000 ppm in males and 1200 ppm (not 6000 npm)
in females after six weeks, in males at 60 and 600 ppm and females a*~ 60
ppm (only) at 15 weeks. The only significant compositional changes in the
leukocytes occurred in males at six weeks when the neutrophils showed a
large increase at 1200 and 6000 ppm, and the lymphocytes showed a small
decrease at the same levels. There were no significant changes in blood
chemistry. Examination of the urine showed lower specific gravity in
6000 ppm males at six and 15 weeks, and 1200 and 6000 ppm females at 15
week;, (all ()-•(> hour spi-c i mens) , ;md in 6000 [>pni I'emales at 15 weeks (16-20
houi :.pecimen) . An i ru iejse in urine vo Limit- w,i., noted in 6000 ppm males
at l'i weeks (both time periods), and in 1200 \>\im I'emales at 15 weeks (0~(>
hour.-,). Or;'..in weight increases occurted in the spleen - 6000 ppm teuiale.s
at two weeks, 1200 anil 6()00 ppm both sexes at six and 15 weeks, and in the
liver - 6000 |>i>iii females at 15 weeks. Relative organ weights showed similar
increases lor the spit-en except for addition ol the male at two weeks/6000
ppm and delation of the male at 15 weeks/1200 ppm; other organs showing
relative changes were: brain - decrease in 15 week males at 600 arid 1200
ppm; liver - increase in two and 15 week females at 6000 ppm; thyroid -
decrease in two week males at 1200 ppm.
-------
The authors could not confirm any relationship between the increased
water consumption at 6000 ppm and the more dilute urine at that level. The
changes in white blood cells and decreased relative brain weights were not
considered related to the dye feeding. The no-effect level of the dye was
considered to be 60 ppm (3 mg/kg body weight/day), with 600 ppm being
reasonably safe.
Yen et al (1971) gave i.p. injections of trypan blue at 50, 100, and
350 mg/kg to rabbits. The lower two doses had no effect on dentin forma-
tion, while the high dose completely inhibited it for eight days.
Tschopp et al (1971) gave i.v. doses of Congo Red, 20-80 mg/kg, to
cats and rabbits. The two higher doses caused an immediate drop in blood
platelets (80-90%), there being no recovery for two hours after the highest
dose, and only partial recovery after the middle dose. The middle dose
had a similar effect on leukocyte count, but recovery was complete in
about 20 minutes, followed by attainment of a long lasting plateau of ex-
cess leukocytes (3 times normal) in 30 minutes. The immediate effect on
leukocytes of the high dose was not clear, but a gradual increase to well
above normal followed. In contrast, in vitro incubation of citrated blood
with Congo Red equivalent to 50 and 100 mg/kg did not affect platelet or
leukocyte count. Remaining platelets showed extensive swelling and pseudo-
pod formation. Within two hours the lungs showed alveolar edema and
capillary obstruction, with larger vessels containing Isrge numbers of
leukocytes and platelet aggregates.
Popa et al (1971) reported that 0.1 mM toluidine blue totally in-
hibited the synthesis of DNA-dependent, highly polymerized RNA in KB cell
culture.
Motoc et al (1971) gave rats 25 mg, orally, of azobisisobutyronitrile
97
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twice a week for three months. Some lesions were found in the stomach,
liver, and kidneys which seemed to be reversible. These correlated in
intensity with decreases in serum glycoprotein and albumin, and increases
in leucine aminopeptidase, glutamic-oxalacetic transaminase, glutamic-pyruvi.
transaminase, glucose-6-phosphate dehydrogenase, aldolase, and 3-glycuronida e.
Iga et al (1971) gave adult male rats 30 umole i.v. doses of the dyes
amaranth and new coccine and studied the simultaneous excretion in bile and
elimination from serum. In four hours 80% of the amaranth had appeared
in the bile (mostly in the initial hour and a half), but only 10% of the
new coccine. The latter was eliminated from the serum at a slower
rate than amaranth: serum half lives were 20 1/2 min. and 5.8 min.,
respectively. The authors examined the ability of the plasma protein to
bind with the dyes, and decided the difference could account for only a
small part of the biliary excretion spread.
Holland and Spain (1971) fed immature male rats 0.06% of 3',N,N-
trimethyl-4-aminoazobenzene for 36 and 66 day periods. Examination of the
shorter period feces for bile acids showed normal amounts of Lithocholic,
deoxycholic, chenodeoxycholic, somewhat lesser amounts of 12-keto-
lithocholic and hyodeoxycholic, and considerably less cholic. The urine
collected from the controls showed the bile acids hyodeoxycholic (day 15)
and lithocholic (day66); the dye-fed animals' urine also showed these two,
but considerably more cholic, hyodeoxycholic and ursodeoxycholic.
Hepatic bile duct oval cells increased from 1% in controls to 41% at
day 46, dropping to 36 by day 53 and staying there. Urinary bile acids
peaked at 26 and (lower) 53 days.
Gafford et al (1971) fed male rats 1-10% of azobisformamide in a low-
iodine diet for up to four weeks, or gave i.p. doses daily for one week of
-------
0.2-20 mg/kg body weight. One day prior to sacrifice each rat was given
radioiodine, i.p. With oral dosing all levels led to reduced iodine up-
take (thyroidal), especially at 5-10% over 10-28 days. Total body weight
decreased about 10% (p<0.02) after one week at 10%. Relative thyroid weight
increased over 20% (p<0.1) after one week at 1%, 40% (p<0.05) after one
week at 10%, and decreased <20% (p<0.001) after 10 days at 5%. Serum pro-
tein bound iodine was lower in the azo fed animals.
In the i.p. dosage the maximum level produced lower iodine uptake (but
only at the p<0.5, 0.9 level). Relative thyroid weignt was unaffected.
Total body weight decreased 10% (p<0.1) at the maximum level in one run,
not at all in another. The authors concluded that permissible levels of
the azo compound in flour are of no concern in thyroid activity.
Gaunt et al (1971) gave immature male and female rats 100-10000 ppm
of Yellow 2G (British food grade) for 13 weeks. No effect on body weight,
food or water consumption was noted. Hematological results were neutral,
likewise serum analyses. Urine examination was without notable findings.
Absolute weights of internal organs showed an increase in kidneys in two
week females at 10000 ppm, a decrease in the ileum of six week males at
1000 and 10000 ppm, an increase in the adrenals of six week females at
1000 ppm, and a decrease in terminal body weight of 13 week males at
10000 ppm - 24 hour fasting was procedure prior to sacrifice and autopsy.
Relative weight changes showed an increase in kidneys of 13 week males at
1000 and 10000 ppm, an increase in the caecum of six and 13 week males at
10000 ppm and 13 week females at 10000 ppm, and an increase in the gonads
of 13 week males at 1000 ppm.
The increase in relative kidney weight was judged to be non-dye
related. The increase in caecal weight has doubtful human significance.
99
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The increase in testes weights was also deemed of dubious significance.
The authors recommended a no-effect level of 1000 ppm (80 mg/kg body
weight/day), or about 2000 times expected maximum intake.
Gaunt et al (1972) gave immature male and female rats 1000-10000 ppm
of Black PN for up to two years. Mortality was considered equivalent to
the controls. Body weight of controls and dye-fed animals was equivalent.
Hemoglobin was lower in 82 week females at 10000 ppm, and 105 week males
at all levels. Packed cell volume was lower in the 1000 and 5000 ppm
groups of these males. Red blood cells were higher in 82 week females at
5000 ppm. Total leukocytes were lower in 105 week females at 10000 ppm.
Serum and urinary biochemical analyses were normal at 52 and 104 weeks.
Absolute organ weights were all normal at 104 weeks. Relative organ
weights were normal except for male liver which was heavier at all levels.
Histological examination of lungs, kidneys, pancreas, liver, and testes
showed no abnormal occurrences in dye-fed animals. Incidence of tumors
in the mammary, pancreas, thyroid, and adrenal glands was normal, likewise
that in the ovary and subcutaneous tissue. The authors recommended a no-
effect level of 10000 ppm (500 mg/kg body weight/day), about 2000 times
maximum expected intake.
Lin et al (1972) gave i.p. injections to rats of 0.3 mmoles/kg body
weight of 4-amino-, 4-methylamino-, and 4-dimethylamincazobenzene. Analysis
of the blood for methemoglobin (as percentage of hemoglobin) showed a rapid
rise to 70, followed by a linear drop to 10 at 7 hours for 4-amino-, a
rapid rise to 50, followed by a plateaued drop to 10 at. 13 hours for 4-
methylamino-, and a slow, irregular rise to 25 at 7 houis, followed by a
linear drop to 10 at 13 hours for the 4-dimethylamino- compound. An in
vitro study failed to generate methemoglobin, implying metabolites were
100
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responsible. Examination of possible metabolites implicated N-hydroxylation
as a highly likely preliminary step.
Singh and Khanna (1972) gave rats a single injection into one testis
of 0.1 ml of a 1% solution/100 g body weight of C.I. Acid yellow 36. Exam-
ination of the testis was conducted at 0-16 hours, 1-15 days. At four
hours there were signs of edema and inflammation. An eight hours both had
increased in intensity and were accompanied by seminiferous tubule degen-
eration, Leydig cell degeneration, and engorgement of blood vessels. At 16
hours the edema had decreased, but now the interstitiam had begun to de-
generate where associated with the tubules also degenerating. After one
day no increase in magnitude of changes had occurred, but in half of the
specimens the seminiferous and interstitial elements had totally degen-
erated. After two days massive degenerative changes showed in the gameto-
genic and endocrine elements. Spermatozoa had decomposed here and there.
After seven days the tubules were totally necrosed along with the inter-
stitial elements. After 15 days the interstitium shows signs of regenera-
tion, but the tubules did not.
Olsen and Hansen (1973) fed male and female pigs 10-160 mg/kg body
weight/day doses of Orange RN for three months. At the highest dose there
was severe hepatic interstitial fibrosis and multiple mdular hyperplasias
of the parenchyma. All dosages caused proliferation of hepatic bile duc-
tule epithelium cells along the triads and the interlobular septa, some-
times intrahepatically also.
101
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IX. ENVIRONMENTAL EFFECTS
A. Persistence and/or Degradation
Mecke, Jr. and Schmahl (1957) reported a study which might have some
bearing on environmental longevity. They incubated a variety of azoben-
zene derivatives with fresh baker's yeast and determined the extent of
decolorization - possibly synonymous with azo cleavage. Azobenzene
did not decolorize, nor did the 2-, 3-, or 4-methyl-, or the 4, 4'-bis(di-
me thylamino)- derivative. Also not decolorizing were phenylazo-2-(N-
methyl)naphthylamine, o-tolylazo-2-naphthylamine, and anisidineazo-2-naph-
thol. The 4-sulfo- derivative only decolorized to the extent of 4%.
In the 10-35% range were 2-methyl-4-hydroxy-, 4-dimethylamino-4'-methyl-,
2-hydroxy-4-dimethylamino-, phenylazo-2-naphthylamine, o-tolylazo-2-
naphthol, and phenylazo-2-naphthol. In the 45-75% range were 2-amino-
azotoluene, 2-hydroxy-, 4-hydroxy-, 3-methyl-4-hydroxy-, 2,4-dihydroxy-,
sodium 4-dimethylamino-4'-sulfo-, sodium 2,4-dihydroxy-2r,4'-disulfo-,
and naphthylazodimethylaniline. In the 80-99% range were 4-amino-,
4-dimethylamino-, 4'-carboxy-4-dimethylamino-, and 2-carboxy-4-dimethyl-
amino-. Solubility in water apparently was not a factor.
Walker et al (1971) removed azoreductases from various rat gut
bacteria and compared their ability to reduce Red 2G. The most proficient
came from Streptococcus faecalis. The following activities relative
to S. faecalis were reported: S. faecalis var. zymogenes-0.95, S.
faecum -0.79, E. coli type 1 -0.62, Proteus vulgaris -0.58, P. mira-
bilis -0.51, P. morganii -0.49, and Staphylococcus aureus -0.02.
-------
B. Environmental Transport
No specific information was found. The azo dyes and foaming
agents investigated are not notably volatile, so their ability to move
once released into the environment is dependent upon water solubility
(and specific gravity for the non-water solubles).
C. Bioaccumulation
No specific information was found. Intestinal bacteria seem
able to destroy the azo bond in many compounds. Some, metabolism tests
indicated "storage" of various azo dyes in skin and fur on prolonged
forced feeding, but to no obvious detriment of the animal.
X. TOXICITY
A. Human-Occupational experience, Other
1. acute, subacute
Hoffman and Guz (1961) on three occasions subjected
themselves to injections of Coomassie blue. On the first, seven doses
over five hours totalled 150 mg in the first three hours and 300 mg in
the last two. On the second, at nine-minute intervals doses of 40,
48, 78, 168, 188, and 276 mg were given; maximum blood level reached
99.5 mg/1. On the third, at nine-minute intervals doses of 40, 36,
72, 161, 186, 194, and 235 mg were given; maximum blood level was 138.5
mg/1. Each time a 2-3 hour period of no effects was followed by 10-15
minutes of ill-feeling, and then fever up to 40°C(104°F), rigors,
hyperesthesia of skin and muscle, nausea, vomiting (blue color), and,
once, diarrhea (blue color). All symptoms subsided after 5-6 hours.
Minor periodic sweating/fever recurred for an additional 2-3 days.
Subsequently, as much as 54 mg in eight hours or 50 ug in 4-5 hours
103
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produced no ill effects In the authors or other subjects. Accidental
perivenous administration produced severe pain after 2-3 hours.
Hb'rstensmeyer (1964) reported a fatal reaction after
i.v. injection of Congo Red in man (mentioned in Tschopp et al [1971]).
Cohen and Bovasso, Jr. (1971) reported on the accidental
ingestion by a 13-month old child of 2500-3000 mg of phenazopyridine-HCl
(Pyridium). Apart from cyanosis of the lips, the only outward symptom
was lethargy. On admission to a hospital within about four hours, the
blood methemoglobin was found to be at least 25%. After another 14
hours this had dropped to 14.6%; normal methemoglobin is 1.7%. Normal
methylene blue treatment for methemoglobinemia proved inadequate, but
transfusion corrected the situation.
2. chronic
No reports were found dealing with chronic toxicity of
azo compounds in humans.
3. sensitization
Meara and Martin-Scott (1953) reported three separate
cases of women developing skin sensitization to aminoazotoluene,
apparently from contact with ball point pen ink containing it.
Foussereau et al (1971) discussed the rather common skin
allergy to Disperse Yellow 3, apparently known for many years. They
showed that any impurities present in commercial material were no more
allergenic than the dye itself.
4. teratogenicity
5. carcinogenicity
-------
6. mutagenicity
7. behavioral effects
No reports were found disclosing these properties of azo
compounds in humans.
B. Birds and Mammals
1. acute, subacute
Zsolnai (1963) reported the following LD-0 and LD-100 doses
in rats after i.p. administration, in mg/kg: 1. aryl-azo-maloni-
triles—phenyl, 5, 15; 2-tolyl, 5, 15; 3-tolyl, 5, 20; 4-tolyl, 5, 30;
2-chlorophenyl, 5, 15; 3-chlorophenyl, 5, 10; 4-chlorophenyl, 5, 10; 4-
bromophenyl, 5, 10; 4-iodophenyl, 5, 10; 4-ethoxyphenyl, 40, 70; 2-methyl-
4-bromophenyl, 5, 15; 2-methyl-4-iodophenyl, 5, 20; 3-methyl-4-bromo-
phenyl, 5,15; 4-methyl-2-bromophenyl, 20, 30; 2-bromo-4-ethoxyphenyl,
40, 60; 2,5-dichlorophenyl, 5, 15; 3,5-dibromophenyl, 5, 15; 2-chloro-4-
bromophenyl, 10, 20; 3-chloro-4-bromophenyl, 10, 20; 2-methyl-4,6-dibro-
mophenyl, 10, 20; 2-chloro-4,6-dibromophenyl, 20, 60; 3-chloro-4,6-dibro-
mophenyl, 5, 20; 4-chloro-2,6-dibromophenyl, 20, 40; 2,4,6-tribromophenyl,
30, 60; 2-nitrophenyl, 5, 10; 3-nitrophenyl, 20, 30; 4-nitrophenyl, 10,
20; 2-methyl-4-nitrophenyl, 5, 10; 3-methyl-4-nitrophenyl, 10, 20; 4-
methyl-2-nitrophenyl, 10, 20; 2-nitro-4-ethoxyphenyl, 10, 20; 2-chloro-4-
nitrophenyl, 5, 10; 3-chloro-4-nitrophenyl, 10, 20; 4-chloro-2-nitrophenyl,
5, 10; 4-acetylaminophenyl, 100, 400; 2-carboxyphenyl, 100, 150; 4-carboxy-
phenyl, 100, 300; 4-carboethoxyphenyl, 20, 30; 3-hydroxy-4-carboxyphenyl,
200, 500; 4-sulfophenyl, 300, 800; 4-sulfonamidophenyl, 200, 500; 4-N-
(4*,61-dimethyl-2'-pyrimidyl)sulfonamidophenyl, 400, 600; 1-naphthyl,
10, 20; 4-bromo-1-naphthyl, 20, 50; 4-phenylazophenyl, 10, 20;
105
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diphenylene-4,4'-bis-(azomalonitrile), 500, 800; 3»3'-dimethyldipheny-
lene-4,4'-bis~(azomalonitrile), 30, 50; 2. aryl-azo-eyanoacetie acid esters-
phenyl/methyl ester, 600, > 800; 4-tolyl/methyl ester, > 800, —; 4-chloro-
phenyl/methyl ester, 200, 600; phenyl/ethyl ester, 400, 300; 4-tolyl/ethyl ui-
ter, > 800, —; 4-chlorophenyl/ethyl ester, 200, 600; 3. aryl-azo-cyanaceta-
nide and Its N-substituted derivatives—phenyl, > 800, —; 4-tolyl, > 800, ,
4-chlorophenyl, > 800, —; phenyl/phenyl, > 800, —; 4-tolyl/phenyl, > 800;
—; 4-ehlorophenyl/phenyl, > 800, —; phenyl/4'-chlorophenyl, > 800, —; 4-
tolyl/4'-chlorophenyl, > 800,—; 4-ehlorophenyl/4l-chloropheriyl, > 800, —:
phenyl/atnino, > 800, —; 4-tolyl/amino, > 800, —j 4-ehlorophenyl/amino,
> 800, —; 4. other—phenylazoacetylacetone, 600, 800; 3-tolylazoacetylacetone,
400, 800; 4-tolylazoacetylacetone, 100, 200; 3-ehlorophenylazoacetylace-
tone, > 800, —; 4-chlorophenylazoacetylacetone, > 800, —; phenylazo-
acetic ester, 100, 300; 3-tolylazoaeetlc ester, 400, 600; 4-tolylazoacetic
ester, 400, 600; 3-ehlorophenylazoacetie ester, 400, 600; 4-chlorophenyl-
azoacetic ester, 200, 300; phenylazodiethyl malonate, > 800, —;
4-tolylazodiethyl malonate, > 800, •—; 4-chlorophenylazodiethyl
malonate, > 800, —.
Niculeseu-Duvaz et al (1966) reported that the LD-50 for
4,4*-dlhydroxyazobenzene and for the 4,4'-bis[-02CN(CH2CH2Cl)2] azoben-
zene was 300-500 mg/kg.
Chadwick et al (1966) reported LD-50's in mice after
i.p. injection, in mg/kg, for; 2,4,6-triamino-5-(4-carbethoxyphenylazo)-
6-N-(2-hydroxy~3-anilino)propylpyrimidine, > 1600; the same, except
chloro in place of hydroxy, 300; the same, except no 2-substituent on
the propyl, 1100.
106
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Sato et al (1966) reported that in immature female rats
repeated injections of N-hydroxy-4-aminoazobenzene in a dose larger than
3.6 mg/100 g proved very toxic, death resulting from methomoglobinemia.
Grasso et al (1968) studied the toxicity of Brown FK
in laboratory animals; chronic results are given in the next section.
Results of the acute study are in Table 33, on rats and mice, 14 and 7
day observation periods, respectively.
Table ?S. Acute toxicity of Brown FK in rats and mice by oral intiibction and
<• intrapcritoneat injection
Repri
Species
Mouse
i
Rat
nted with
Cosmet. Toxicol
Route
Oral
Intrapcritoncal
Oral
Intrapcritoncal
permission from
. 6:1-11 (1968)
Single
dose
(g/kg)
2-0
1-0
1-5
2-0
2-0
4-0
8-0
0-75
1-15
1-69
Food
.
Deaths/group of
5 animals
Male
0
0
2
5
0
1
1
0
4
5
Female
0
0
1
5
0
0
1
_ •'
_
-
Copyright by Pergamon Press Ltd.
Autopsy failed to reveal the cause of death.
Gaunt et al (1969) reported oral LD-50's for Ponceau 4R
in rats and mice of >8 g/kg, and i.p. LD-50's of 1.6-1.9 g/kg in mice
and 2.6 g/kg in female rats; male rats had about the same i.p. LD-50 as
females, after 48 hours, but after seven days the male value dropped to
0.6 g/kg (all values taken from a 1967 publication by Gaunt et al).
On autopsy, renal tubular necrosis was found. In 1957 a German report
id"
-------
quoted LD-50 values for rats of 2 g/kg i.p. and 1 g/kg i.v.
Gaunt et al (1969 pp. 557-563) reported oral LD-50's
for Black PN to be greater than 5 g/kg in mice (1957 German report) and
also in rats (1967 Gaunt et al). In the earlier Gaunt report i.p.
LD-50's of 0.5-1.0 g/kg for mice and 0.9-1.2 g/kg for rats were also
disclosed.
Caldwell et al (1971) reported minimum lethal oral doses
in mice for some azo derivatives of the 3-tropanyl (R) ester of 2,3-
diphenylacrylic acid (in mg/kg) : 256 for [CeH5CH = C(C02R)C6Hif-m-N^2»
768 for [C6H5CH = C(C02R)C6Hl+-p-N^2, 768 for CgH5CH = C(C02R)C6Hl+-m-N =
N-C6H5, >1024 for C6H5CH - C(C02R)C6Hlt-p-N=N-C6H5, and >1024 for
C6H5CH = C(C02R)C6H3(-p-OH)-m-N = N-CgH5.
The Toxic Substances List-1973 Edition provided the
following compilation of acute toxicities, including the year of publi-
cation of their source. LDLo, TCLo, and TDLo are their abbreviations
for lowest published lethal dose, toxic air concentration, and toxic
dose, respectively. The parenthetical chronologies refer to obser-
vation periods.
Acid Red 26 LD-50, mice, i.p., 2 g/kg, 1966
4-Aminoazobenzene (AAB) LDLo, mice, i.p., 200 mg/kg
TDLo, frogs, i. renal, 110 mg/kg, 1964
4-Amino-N,N-bis(2-chloroethyl)-2'-carboxy-2-methylazobcnzene
LD-50, rats, i.p., 20.2 mg/kg, 1964
4-Amino-N,N-dimethylazobenzene (DAB)
LD-50, mice, i.p., 500 mg/kg, 1962
TDLo (1 week), mice, oral, 5 mg/kg, 19^8
TDLo (40 days), rats, oral, 800 mg/kg, 1956
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4-Amino-2'3-dimethylazobenzene
TDLo, mice, s.c., 330 mg/kg, 1965
4-Amino-N,N-dimethyl-4'-fluoroazobenzene
TDLo(12 weeks), rats, oral, 3.2 g/kg, 1953
4-Amino-3',5'-dimethyl-4'-hydroxyazobenzene
LD-50, rats, i.p., 350 mg/kg, 1963
TDLo, rats, i.d., 100 mg/kg, 1963
4-Amino-4'-hydroxyazobenzene
LD-50, rats, oral, 1.95 g/kg, 1963
LD-50, rats, i.p., 300 mg/kg, 1963
4-Amino-4'-hydroxy-2,3',5'-trimethylazobenzene
LD-50, rats, i.p., 142 mg/kg, 1963
LDLo, rats, oral, 600 mg/kg, 1963
TDLo, rats, i.d., 100 mg/kg, 1963
4-Amino-2',N,N-trimethylazobenzene
TDLo (6 weeks), rats, oral, 1.5 g/kg, 1969
Azobenzene LD-50, rats, oral, 1 g/kg, 1966
Azobisisobutyramide-HCl
LDLo, rats, oral, 400 mg/kg, 1971
Azobisisobutyronitrile (AIBN)
LD-50, rats, oral, 700 mg/kg
LDLo, mice, i.p., 25 mg/kg
Azoethane TCLo, rats, inhal., 4800 ppm/hr, 1968
l,l'-Azonaphthalene TDLo, mice, s.c., 200 mg/kg, 194C
l,2'-Azonaphthalene TDLo, mice, s.c., 200 mg/kg, 1940
Congo Red LD-50, rats, i.v., 190 mg/kg
Food Brown 3 LD-50, rats, i.p., 375 mg/kg, 1966
Food Red 3 LD-50, rats, i.p., 1.1 g/kg, 1967
Food Yellow 3 LD-50, mice, i.p., 4.6 g/kg, 1967
(FD&C Yellow 6) LD-50, rats, i.p., 3.8 g/kg, 1967
Solvent Red 24 TDLo, rats, s.c., 8.32 g/kg, 1958
Solvent Red 80 TDLo, mice, i.p., 80 mg/kg, 1968
Trypan Blue LD-50, mice, i.v., 267 mg/kg, 1970
LDLo, rats, i.v., 300 mg/kg
JOV
-------
2. chronic
Metcalf (1962) gave adult rats three s.c. doses of 20 mg
of Evans Blue and Trypan Blue on alternate days (separate experiments
for each dye). This treatment resulted in death of the rats in three
weeks. The i.v. LDLo for Trypan Blue in rats, above, corresponds to 60
mg per 200 g rat, an interesting, though not directly comparable,
comparison.
Davis and Fitzhugh (1963) fed male and female rats
0.01-1.0% of D&C Red No. 10 for two years. There was no effect on growth.
There was a definite iWrease in longevity at the 1% level. Survivors'
splenic weight (relative) increased 2-3 times in both sexes at the 0.25
and 1% levels. Slight to moderate bone marrow hyperplasia was noted in
the 0.25% females and both sexes at 1%. Frequency of occurrence of the
wide variety of tumors found was not significantly different from that
in the controls.
Oser et al (1965) treated for two years male and female
dogs and rats with a diet composed mainly of bread (overall nutritionally
balanced for each animal) which had been made with 100 ppm of azobis-
formamide. This level was ten times the proposed use level, about twice
the maximally permitted level, and was all that could be incorporated
without interfering with the baking process. There were no adverse
effects on the original animals, or on three subsequent generations of
rats.
Chadwick et al (1966), in an anti-tumor study, gave rats
i.p. doses on five successive days of 2,A,6-triamino-5-(A-carbethoxy-
phenylazo)-6-N-(3-anilino)propylpyrimidine, and its derivatives with
110
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chloro or hydroxy substitution on the 2-propyl position. They found
approximate LD-50 values of 550 mg/kg for a 400 mg/kg/day dose for the
parent compound, 140 mg/kg for a 100 and 50 mg/kg/day (not clear in the
paper) dose for the 2-chloro, and >400 mg/kg for a 400 mg/kg/day dose
for the 2-hydroxy.
Ikeda (1966) fed rats 0.2-5.0% of Ponceau MX for up to
15 months. Mortality was the same as the control group. At 1 and 5%
growth was noticeably retarded even though food consumption was normal;
water consumption in these two groups increased after the eighth
month. All levels produced heavier than normal livers and thyroids from
at least the third month. The kidneys were heavier at the 1 and 5%
levels. Liver cell adenomas were seen in dead animals as early as 10
months, the incidence increasing with amount of dye fed. Obvious renal
tubular degeneration had occurred by three months at even the 0.2% level.
By the 15th month all groups (including the control) showed glomerulo-
nephritic and nephrotic changes, also interstitial cell infiltration.
Incidence and severity were greater in the dye-fed animals than the
controls.
In an in-progress study on feeding the same range of this
dye to mice for up to 12 months, liver tumors were found at the 0.2%
level.
Grasso et al (1968) did a chronic toxicity study on
various laboratory animals of Brown FK and some of its isolated azo
components. Rats and mice were given in their diet for up to 43 days
0.1-2.0 g/kg of Brown FK, or by i.p. injection 43 doses of 0.1-1.0
g/kg. Guinea pigs, hamsters, and rabbits were given sto*nach tube doses
111
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of 1 g/kg Brown FK for up to 14 days. Rats received up to 16 daily
doses by stomach tube of the FK components 2,4-diamino-5-(p-sulfophenyl-
azo)toluene (I), l,3-diamino-4-(p-sulfophenylazo)benzene (II), 2,4-
diamino-3,5-bis(p--sulfophenylazo)toluene (IV), or l,3-diamino-2,4,6-
tris(p-sulfophenylazo)benzene, a 40/60 mixture of I/IV, or a mixture of
II, VI, l,3-diamino-4,6-bis(p-sulfophenylazo)benzene (III), and 1,3-
diamino-2,4-bis(p-sulfophenylazo)benzene (V); the dose was 0.5 g/kg.
Similarly, 1 g/kg doses were given of 50/50 I/II, 40/60 I/IV, and
I/II/III/V/VI (50% I).
The 1 g/kg i.p. doses of FK had no effect on mice. The 2
g/kg oral doses (six) had no effect on mice. After 28 oral 1 g/kg
doses, one out of twelve mice showed heart and skeletal muscle changes.
Both sexes of two strains of rats died after receiving 3-8
doses (stomach tube) of 1 g/kg of FK. The observed symptoms were,
sequentially, growth retardation, heavy weight loss, lethargy, piloerec-
tion, hypothermia, difficult breathing, death. Sacrifice of the animals
just after weight loss was noticed revealed greyish-white areas in the
ventricular myocardium, conjested liver and lungs. Lesions were found
in the heart and skeletal muscles.
Of the rats given 1.5 g/kg of FK, one-third developed centri-
lobular hepatic necrosis and severe fatty change.
The following tables condense the muscle damage information
obtained from stomach tube dosing.
112
-------
Tr.'}!c< 31, Incidence of strut "d-ninsclc damage induced by fiiowit FK in relation to dahy dose administered by
stomach lube to ruts
M.- K...
0 0 2 -
0-1 .<>-
V 1
(_' *
1 • ;
..
1-S
?..,
O, ••-••;:-.- , v, T
Table 3f, l.-.cuL-
Percentage of rats affected
.._ r.lts SkcieM'
i e N'c in fx. m:r
60
•13 2o 20
. ! VI
I,
i!
S "• 10
6 ''. 10
c 10
- ,v "V -u.i -. Joscs ,ibov: !
1
: '!-y-
H.-.-it
0
10
ir'
/j.
JO
90
90
Tor.j',1
0
10
0
3J
07
—
—
_
:c mi
isc'c Din
0
0
10
33
52
—
—
--
phu:gni
0
10
0
22
65
—
—
—
•;/rc ofstiic.icd-nutsclc damage induced by components of Brown f-'K administered by stomach
tube
/o rats
Percentage of rats affected
Mm
rdrn n
^aiinc
r,ro\vn.'-K.
Comp< ."id I
Compound 11
Compounds 1-!
CompiMiids I-r
Compounds I r
Conip.'.ind IV
Compound VI
Compounds IH
Comp.'i.inds I-i
Daily
:crhl dose
i-.tcK-d (fi/kp,)
—
1-0
0-5
0-5
III 1-0
1VJ; 0-5
IV J 1-0
0-5
0-5
-1I1 + V+VI 0-5
I! J III i V-f-Vlt 1-0.
No. of
doics*
6-16
2-16
2-6
2-6
2-3
6
3-16
16
7-12
6
5-6
No. of
'rats
examined
20
16
9
10
12
10
• 10
10
6
It
10
Heart
0
69
100
30
75
0
10
0
0
0
50
Tongue
0
94
89
50
100
0
30
0
0
0
70
Skeletal
muscle Diaphragm
0
75
22
30
100
0
0
0
0
0
40
0
81
Tongue
12
59
64
0
0
Skeletal
11 HI sue
65
59
, 55
0
0
•Diapiir;'rm
62
75
82
0
0
I;or each species, juoiip^ of similar size \\erc dos_\i willi v..,!er and no !, .ions wi-ic si.cn.
Tables 34-36 reprinted with permission
from Food Cosmet. Toxicnl. fi-i-ii (1968)
Copyright by the Pergamon Press Ltd
113
-------
The i.p. dosing was more toxic. Although the 0.1 g/kg
regimen was well tolerated, the 1 g/kg level was usually fatal after
10 shots. Only three 1 g/kg shots sufficed to greatly reduce splenic
weight, and increase relative renal weight. The renal tubules were
dilated, vacuolated, and pigmented with FK (not pigmented at all after
oral dosing). The i.p. dosing did not produce the muscle damage seen
from oral dosing, indicating metabolites were probably responsible. It
was suggested that, since some animals were unaffected by even the
highest doses, there was intraspecies variation in the gut microflora
apparently responsible for the metabolizing of the azo linkages.
Grasso and Golberg (1968) reviewed the rather extensive
literature on metabolism and toxicity of Brown FK, using some lessons
learned to comment on the nature of the testing procedures for food dye
toxicity.
Gaunt et al (1969) fed male and female pigs Ponceau 4R
at 100-900 mg/kg body weight/day for 90 days. There was no effect on
weight gain. Hematological findings were normal at 300 mg/kg/day. At
900 mg/kg/day males showed reduced hemoglobin and red blood cells at
week six but not at week 13. Urine and serum chemical analyses were
normal. Relative internal organ weights were also considered normal.
Autopsy and histopathology on sacrifice of the animals revealed no
\
abnormalities.
Grasso et al (1969) fed male and female rats 0.125-1.0%
of Ponceau MX (also known as 2R) for up to two years. Fatalities became
statistically significant at P < 0.05 by the 88th week in both sexes
at the 0.5% level. However, by the 80th week all levels in males and
-------
0.25% up in females showed somewhat higher mortality. The 0.125% level in
females never did produce unusual mortality. Body weight increase showed
a slowdown in females at 1% by the 3rd week, at 0.5% by the 25th week, at
0.25% by the 51st week, and at 0.125% by the 65th week; there wasn't any
noticeable reduction in the weight of females from a peak weight. In males
initial slowdown began later, by week 39 at the 1% level, by week 65 at
levels 0.25 and 0.50%, and by week 91 at 0.125%; males showed sharp, con-
tinuing drops in weight between weeks 65 and 77 at levels 0.25% up, and
between weeks 65 and 91 at 0.125%. Examination of food eaten at consecu-
tive three-month intervals showed decreases by females only over the three
intervals covering weeks 54-93 at the 0.5% level; males showed decreases
over the 94-99 week period at 0.125%, 80-99 weeks at 0.25 and 0.5%, and
66-99 weeks at 1.0%. The animals didn't care for the taste of the dye
even at the 0.125% level, but the palatability showed a very high decrease
going from 0.5 to 1.0%.
Hematology revealed decreases in hemoglobin in males by week
14 at the 0.5 and 1.0% levels and in females at the 1% level; by week 29
females were showing decreases at the 0.25% level and w«re also showing
decreases in hematocrit at that level and up. By week 61 males were
showing lower hemoglobin at 0.125%, lower hematocrit at 0.25%, and lower
red blood cells at 0.25%. At week 79 only hemoglobin was low at the
highest level in both sexes, except that males showed a higher total
leukocyte count at 0.125%. At week 104 hemoglobin was low in both sexes
at 0.25%, also hematocrit; in males the red blood cells were also low at
that level, also total leukocyte count. Chemical analysis of the blood was
normal after two years. Urine analysis showed higher ascorbic acid after
one year at 0.25% up, otherwise being normal chemically and physically at
-------
one and two years. Relative internal organ weights after two years showed
increases in the brain of both sexes at 0.5% (no 1% values for any organs
were determined because of too few survivors), heart of males at all le-
vels, liver of both sexes at all levels except 0.125 in females, kidneys
of both sexes at all levels, adrenals of both sexes at 0.5%, and pituitary
of males at 0.125 and 0.5%. Incidence and severity of pathological
hepatic changes were directly related to dose level, but there was nothing
to suggest carcinogenicity.
Gaunt et al (1969) fed male and female pigs Black PN at 100-
900 mg/kg/day for 90 days. There was no effect on body weight. Hematology
was normal. Relative internal organ weights were normal. There seemed
to be evidence of ileal irritation at 300 and 900 mg/kp/d, possibly re-
sultant from the feeding of the daily dose in a highly concentrated form.
Gafford et al (1971) gave male rats daily i.p. doses of
0.2-20.0 mg/100 g body weight of azobisformamide. The high dose caused
62% mortality in one week, starting on the third day. The deaths were
preceeded by 1-2 days of bloody urine.
Galea et al (1972) gave rats 30 mg/d of Amaranth for up to
545 days, as 0.12% of their diet. Between 60 and 180 days weight began
to lag behind the controls, but the absolute difference didn't in-
crease between 180 and 360 days. Apparently there was considerable
mortality in the dosed animals, but inconsistencies in the paper obscured
the results. Hepatic vitamin A was only half that of controls at 60 days,
20% at 180 days, and 0-10% at the end of the experiment.
Shtenberg and Gavrilenko (1972) fed male and pregnant female
rats a daily dose of 1.5 mg/kg or 15 mg/kg Amaranth for 12 months.
116
-------
At the end of the test period both levels had about the same ability to
reduce spermatosoidal.resistance, depress the estral cycle, and heighten
the gonadotropic function of the hypophysis. Postimplant. mortality was
far higher, 3-5 fold. The number of live births per rat dropped 30%.
The fetal and placental weights were slightly lower at 15 mg/kg. The
1.5 mg/kg level was the maximum recommended at the 8th FAO/WHO session
on food additives.
Collins et al (1972) gave pregnant rats 7.5-200 mg/kg/day
of Amaranth during days 0-19 of gestation. They found no change in the
number of corpora lutea, nor any adverse effect on implantation. Fetal
mortality increased notably at a dosage over 15 mg/kg. Weight of live
young was not affected. Percentage of resorptions increased with dose.
Skeletal and soft-tissue abnormalities were not dose related.
The Toxic Substances List-1973 Edition provided the follow-
ing compilation of chronic toxicities; see the last entry under Section
X.B.I, for elaboration of terms.
Acid Red 26 TDLo, rats, oral, 62 g/kg/65 weeks, 1966
Acid Red 148 mice, s.c., 80 mg/kg/4 weeks, 1950
(intermittent dosage)
4-Amino-N,N-dimethylazobenzene (DAB)
TDLo, hamsters, oral, 9.6 g/kg/42 weeks, 1961
(intermittent dosage)
4-Amino-2,4'-dimethylazobenzene
TDLo, mice, oral, 28 g/kg/595 days, 19*9
4-Amino-2,3'-dimethylazobeneene
TDLo, mice, oral, 28 g/kg/595 days, 1949
4-Amino-2',3-dimethylazobenzene
TDLo, hamsters, oral, 25 g/kg/49 weeks, 1961
TDLo, mice, oral* 2.8 g/kg/70 days, 1955
117
-------
4-Amino-3,4'-dimethylasobenzene
TDLo, mice, oral, 28 g/kg/595 days, 1949
4-Amino-3',5'-dimethyl-4'-hydroxyazobenzene
TDLo, rats, oral, 1000 ppm/2 years, 1963
4-Amino-4'-hydroxyazobenzene
TDLo, rats, oral, 700 ppm/2 years, 1963
Food Red 1 TDLo, rats, oral, 1.2 g/kg/day, 1953
Food Red 6 TDLo, rats, oral, 182 g/kg/2 years, 1963
Food Yellow 3 TDLo, mice, oral, 35.4 g/kg/52 weeks
(FD&C Yellow 6)
Solvent Yellow 14 TDLo, mice, implant, 80 mg/kg* 1968
Trypan Blue TDLo, rats, oral, 440 mg/kg/21 days, 1958
(intermittent dosage)
TDLo, rats, s.c., 1.088 g/kg/87 weeks, 1963
(intermittent dosage)
3. sensitization
Gordon (1964) tested the correlation of carcinogenicity
with skin sensitization in a group of butter yellow derivatives. The
results are in Table 37. DAB is an abbreviation for 4-amino-N,N-dimethyl-
azobenzene, and MAS is DAB minus one methyl. In column 2, the higher
the number the higher the carcinogenicity in rats (not necessarily
the same in the guinea pigs used here). The cross-reaction test was
run at the same time as the confirmation of sensitivity test; it
indicated that an animal sensitized by one of the azo compounds was
likely to be sensitized by all. Reaction to the sensitizing compound
itself ranged from 100% from a 1% solution of the confirmation dose,
to 18% from a 0.001% solution. The desired correlation was not
achieved.
118
-------
Reprinted with permission from Nature
203:884-885 (1964). Copyright by
MacMillan Journals Ltd.
T:lbIi'J7, SENSlriZI.VU CAPACITY _IM> C.LHCINOGEMUTY Or AZO DYES
A /.c,
iil «' *
1)AU
3'(.'II,I>A11
:IT DAI;
4'CU,DAi'.
4'F DA L!
-UAli
2 CHjDAti
•i'Mr.DAI!
3'Ct',l).Mt
2 Oil DAB
2'OH DAB
jj-Ammonzo-
benztne
Azobenzene
Carnnn-
genic *
Ind-'v
(rat)
6
SO-12
10-12
< 1
10-12
6
0
0
0
0
0
0
0
Bliuliiut
t« rat
liver
protcim
-r
-r
T-
+
4-
-
T
0
0
a
0
a
0
Capaeuyj
to
aensitl/c
100 (4)
joo (;;
100 (5)
100 (5)
100 (5)
40(0)
100(4)
0(J)
0(5)
0<5)
0(5)
0(5)
0(2)
Cnp,icit>
to I'l'CIt
skin
reuctl.ii:>:
100 (j)
r j <-t>
OC (i:>)
73 ilS)
80 (IS)
.i.) (13)
CO (5)
75(8)
50 (20)
10 (20)
10 (JO)
5 (20)
0 (12)
• For furnaii.c sco text.
t Si-u ii'l. 2.
t The Tiumbcrs refer to the jicrcem.i'4! of animals aeniitucd; those in
brackets lutlic.ite the uurnber.s of aunn.Us in cjieh experiment.
§ Cross-rfiu ttons: the nuinbers rct>r to the porceiitatieot anininla reacting;
th.me m brjekcts mdicuto cho number of scusiuzcd aniinalD skin tested.
4. teratogenicity
(Kelley et al, 1964, quoted T. Vickerstaff, 1954, from
The Physical Chemistry of Dyeing: "It is virtually impossible to
synthesize a pure disazo dye and only slightly easier to purify a
given sample." Most of the papers in this section deal with trypan
blue, a disazo dye!)
Hamburgh (1952) injected female mice with about 1.7 mg
trypan blue one week prior to, and one week past fertilization.
Nearly 1/4 of the young showed tail abnormalities. Of these malformed
young, 3/4 were males. The experiment was repeated, but the mice
were sacrificed 10-14 days after fertilization. There were abnormalities
in 60% of the embryos, mostly in the tail and head regions (everted
brains). The latter probably resulted in failure to survive to term.
Waddington and Carter (1953) injected female mice in
the seventh day of fertilization with 5 mg of trypan blue. In one
experiment the young were allowed to be born, in another, the mothers
119
-------
were sacrificed at one day intervals after injection. Mortality
(embryo) rose sharply on the llth day of gestation. Abnormalities were
notably high one day after injection, but decreased sharply on the
llth day. Of the full term animals, litter size was half the normal
and males predominated. Head and tail abnormalities predominated.
Hamburgh (1954) reported on a follow-up study in which
pregnant mice were injected with 5 mg of Trypan Blue on the 7th day
of gestation, and then sacrificed on the 8-14 days of gestation. He
found 15% resorption and 60% malformed embryos, mostly head and tail
types. The chronology of the malformations is presented in Table 38.
TAB UK 38,'
Incidence of malformation of embryo* 8$~H days after fertilisation from mother injected with trypan blue
J^ DAVb
8J- 9
0-05
m-io
105-11
lli-12
12J-13
13J-14
Total
TOTAL Or „„„„,,,
IttftCYOa >OF *C8
43 19
r,s o
KI ?:>
RO !l
08 10
JfiiJ -50
S3 20
571 164 '
23
41
41
fll
«2
76
28
332
1
8
17
4.»t
20'»"
20*""
5""
75" '
rowuror
UAUTI>,§
ft
14
13
in
22 -
2"
8
106
%OF1>F.AD MnI?"?'',Cr fcOTTAIL MITO.
HAUT1E8 VAt'TIM M*MTI«8 MAUT1B8
£0 . . . , 13
25 ... , . . 34 ,
10 ., ,, 27
If, ' 4,1 A3 34
22 41 42 41
17 S? 23 23
15 24 45 9
18 140 20 191
% or MISC.
**'•'«*•
53
01
33
no
41
14
Ifl
33
'In breaking down abnormnlitici into the various categories ninny cnsee were Hated more tlwn once, whenever more than
one type of malformation occurred in the iaitie embryo. Thi» fact should be borne in mind whfa adding the flgurci of table 1.
"Records of rcsorbtion ore incomplete after the 10th doy. Reprinted Wl'th permission frOHl Andt ReC
119:409-27 (1954). Copyright by'WistaF^
Institute Press.
Wilson (1955) gave s.cT injections of 10 mg of a variety
of trypan blue-related azo dyes to pregnant rats on the 7th, 8th» and
9th days of gestation. On the 20th day all were sacrificed. Table
39 contains a list of the dyes (o-toluidine is a non-azo ingredient
120
-------
Reprinted with permission from Anat. Rec.
123:313-333(1955). Copyright by"
Wistar Institute Press.
TABLE 3
12
1
' Specified in teit.
cotmnon to the manufacture of the others), the mortality, and terato-
genicity found; the mothers had been surgically examiaed for the number
of implants prior to injection, on the 7th day. Table 40 breaks down
the malformations according to most frequent type. In the last column
of this table, "other" consisted of gastroschisis, short snout, and
clubfoot, about equally. The most frequent brain effect was hydrocephalus
121
-------
accompanied by mesencephalic aqueductal obliteration/constriction.
Ocular effects included monolateral (usually) anopthalmia, microphthalmia,
and retinal coloboma. Cataract of the lens was also seen, and deemed
to be degenerative rather than developmental in nature; its incidence
was not known as few animals were examined for it. Cardiovascular
effects were (decreasingly): aortal-pulmonary trunk transposition,
aortal right-sided arch, aortal double arch, absence of ductus arterio-
sus, and trunkus arteriosus communis. Vertebral colunnar effects
were rudimentary-absent lumbar, sacral and caudal vertebrae, absence
of entire sacrum, medially displaced ilia. Resultant external and
internal changes included trunk shortening, lack of tail, lack of
genital/excretal openings, lack of some pelvic viscera.
There was no evidence for the dyes having crossed
the placenta. This, together with the occurrence in the same litter
of normal appearing (inside and out) embryos at term, did not allow
for even an intelligent guess as to the reason for the malformations.
Langman and van Drunen (1959) injected s.c. female rab-.
bits with 5 ml/kg of a 1% trypan blue solution five days prior to,
two and seven days post-fertilization. The uterus was excised on the
28th day of gestation. Serum proteins were analyzed at pre- and post-
fertilization intervals. Table 41 contains the overall teratogenic
statistics. Malformations were found in the spine, tail (none), gut
(eventrated), and brain (hydrocephalus). The eyes were normal. Control
rabbits showed a decrease in total serum protein and albumin during
gestation, but dye-dosed ones showed an increase in both, especially
during the first 14 days, followed by a decrease to non-pregnant levels.
122
-------
The y-globulin fraction was not increased.
TABUS ti
nu fl ( lining flul)
|Hr trut rru
138
*0
128
102
17«
31
0
? 1
3 i
2
1 '
0
12
Reprinted with permission from Nature
187:605-7 (I960). Copyright by
MacMillan Journals Ltd.
-------
Beaudoin (1961) injected, at 36 hours' incubation, 50 yl of 0.1% dye
solution into the subgerminal cavity or 100 yl of 0.1% dye solution
into the yolk sac of chicken eggs. Embryos were sacrificed on 10th day.
The same was done using trypan blue at 0-96 hours of incubation to test
the time span effectiveness of the teratogenicity. The dyes tested
in the first study and results are given in Tables 43 and 44.
TABLE U3-
Teratogenic activity of several disazo dyes on chick development when
injected at the 36th how of incubation
Dye
Trypan blue
Fvans blue
Niagara blue 4B
Niagara blue 28
A?o blue .
Congo red
Saline controls .
Untreated controls .
Total treated
Sub-
germinal
101
78
94
67
82
95
220
—
Yolk
sac
94
90 *
93
86
93
90
141
134
Percentage
mortality
Sub-
germinal
45-5
44-8
69-3
23-9
18-3
59-0
20-9
—
Yolk
sac
56-5
14-4
299
93
107
33
12-0
11'9
Percentage malformed survivors
Siib-
germinal
72-8
jfj
%4-S
27-4
20-9
35-9
201
—
P values*
< 0-001
<000l
0 !fl
0-47
0-90
0-08
—
—
Yolk
sac
56-2
5-2
33-4
0
0
3-4
2-4
3-4
P \alues
< 0001
0-52
<0001
—
—
098
—
—
' P values derived from X1 test for independence.
TABLE Ui.
Frequency of malformations among surviving 10-day chicks
, (percentage of total survivors)
Dye
Tr> pan blue .
Evans blue
Niagara blue 4B
Niagara blue 28
Azo b!u£
Congo red
Saline controls
Untreated controls .
I
Rumples*
SO' YSt
69 I 53 6
51-2 2-6
206 31-8
196 —
14-9 —
334 23
96 24
— 2-5
lye
SG YS
7-3 2-7
7-0 1-3
17-2 3 4
19 —
104
12 8 2-3
4-6 0 8
—
Beak
SG YS
109 —
2-3
17-2 —
— —
7-5 —
7-7 —
15 08
— 0-8
Ga>lroh
SO YS
fttrtct limb
SG YS
36 — 171 A B
4-7 —
34 1-5
15 —
26 —
4 I —
— 08
9 3 1-3
34 —
1-5 —
2-6 —
1-5 —
Spina biftJa
SG YS
_
47 —
__
39 —
1-5
7-7 —
8-6 08
Other
SG >S
— 24
— 6-J
— 45.
39 —
15 —
t 5
_ _
Tables 43 and 44
reprinted with permis-
sion from J. Eiabryol
Exptl. Morphol. 9, pt.
1:14-21 (1961). Copy-
right by Cambridge
University Press.
SG = subgcrnim.il.
t YS =•- yolk sac.
The eye defects were anophthalmia and microphthalmia; beak defects
were cross-beak and small beak. The separate trypan blue study indicated
insignificant mortality after 48 hours from eubgerminal, and 72 hours
12).
-------
from yolk sac Injection. Percentage of malformed survivors was unaf-
fected after 72 hours from subgerminal or yolk sac injection. The
percentage of malformations regardless of survival peaked at 36 hours
after yolk sac, and at 36-48 hours after subgerminal injection.
Beck (1961) obtained three commercial samples of trypan
blue and made up solutions of each, 0.01 mole/ml (confirmed by titra-
tion of the azo linkage). Pregnant rats were given s.c. injections
at 8 1/2 days of gestation of 0.05 mole azo linkage/kg. They were
sacrificed at 20 1/2 days of gestation. One of the samples of trypan
blue failed to produce any abnormalities, and caused a statistically
insignificant increase in percentage of resorptions. The other two
samples produced resorptions and abnormalities typical for this rat
strain, but in differing amounts. An attempt to see if there was a
difference in toxicity between the samples by determining the LD-50
values on non-pregnant females somewhat equivocally indicated that
the non-teratogenic sample was also less toxic than the one of the
others which was used for comparison.
Hoar and Salem (1961) gave a single injection of 2 ml
of 1% trypan blue s.c. to pregnant guinea pigs on a day selected from
days 6-13 of gestation. The litters were sacrificed or. day 30 of
gestation, when it was expected that all organ formation would have
been completed (term is 68-70 days normally). There wasn't any
pattern during this time span to the reduced weight and reduced crown-
rump length seen. Resorptions were highest when the dose was given
on day 12 or 13, next highest on day 7. Gross malformations were
highest when the dose was given on day 11, day 9 being next. Half of
12$
-------
these malformations were cysts of the anterior thoracic wall, one-third
were spina bifida; in decreasing amounts, also seen were microphthalmia,
hydrocephaly, edema, and meningocele (all <6%). About half of the
small or otherwise abnormal embryos had a posterior cleft palate (pos-
sibly merely related,to delayed growth).
Some of the gestations had been allowed to go to term.
Approximately l/6th of these resulted in complete resorption, and
another l/4th aborted. The gestations were about one day longer than
controls and litter size was smaller (though considerably heavier).
These had only 5% abnormalities, all non-fatal.
Izumi (1962) injected six azo compounds, of varying
carcinogenicity, into pregnant mice to study the teratogenic effects.
There was no apparent correlation of carcino- and teratogenicity.
Tables 45-61 present the findings.
Table 15". The development of'the fetuses in the control groups with or without
intrapentoiieal injection of 0.01 ml peanut oil per g of body weight
I pay of Total Total Fetuses at the 18th day of gestation ; No. and
Group injecti- No. of : No. of (
-------
Table %• Effects of 3'-trifluororaethyl-4-dirnethylamincazobenzene on the offspring of mice
Clntraperitc^eai injection of 0.01 ml 0-3 peanut oil solution (0.3 mg) per g of
body weight]
' Day of Total ' Total
Fetuses at the 18th day of gestation
mot he- irnplan-j survived , resorbed ; dead | survivor^ body weight :
| malform- mean+stand-
i | ed ard error
107 jj-l (31.8) |73 (68.2) j 0 ( 0 ) I 1 ( 2. 9) '• 0.88x0.015 IDt
injecti-
on
8- 9
M- li
i .'-;.)
14 -10
No. of
mothe-
rs
No. of
implan-
tations
1-1 j 107
2i
18
149 '
114 i
"
11 70 ,
__ '• No. and types ; of
f ~ I each anomalies
149 rS (.51'. 3) ;ri (.47.7) , 0(0) Jj (1H.2) J O.y7+0.0!8 .ijV
114 157 (50 0) (37 COO 0) | 0(0) '14 Ci'4. C) < 1. 03±0. 018 ,12Cp. ]Ct. 2Dt
T
0 ,61 (91.4) ; 6 ( 8. o) : 0(0)17(10.9; 1.04+0.018 ; 7Cp ,
*• An: matforniation of ankle jomt, Cp: .cleft palate. Ct: curved tail, Dt: deviation of
toe. or finger. El: malformation of elbow joint, Kn: malformation of knee joint, Md.
macrodactyly of toe, Pd: polydactyly of toe.
x : "i of total No. of implantations, £\ : °a of survived.
Izumi (1962) reported a study which concentrated its ef-
fort on changes in fetal bone structure as a result of these dyes
being injected into pregnant mice. Tables 62-91 present the results.
Table Ml- Effects of moncethylaminoazobenzene on the offspring of mice [Intraperitoneal
injection of 0.004 or 0.006ml a°a peanut oil solution (O.I1 or 0.3 mg) per g of
body weight]
I Day of j Total j Total ;
No. of j No. of ;
Fetuses at the 18th day of gestation
_ _ __ _____ ___
0~x^ TvTxT <<•/,) x. ~ (°e) 1 ( .'.5; j 1. 0:'±C. Oi'O i 1 Kn
i 10 -a
11'!) 73 f.:6. 6; 55 (42. G) 1 ( 0.8) ' 5 ( 6.8) 1.07±0.018 i 3 Dt, I Kn. 1 Pd
OS 3H (Cl P) ^3 (39.0) 0(0) . 1 ( 2.8) ' 0.97±0.033 1 Dt
44 .30
14 (31.6, ' 0 ( 0 ) : 1 ( 3.3) ; 0. 9l±0.029 1 An, 1 Kr.
* An: maJformation of ankle joint, Dt: deviation of toe or finger. Kn: malformation of knee
joint, Pd: polydactyly of tee. x : °', of total No. of implantations, ZA : ?J of survived.
Table IS- Effects of 4'-methy!-4-dimei.hy!aminoazobcnzene on the offspring of mice
(Intraperitoneal injection of 0.01 m! o'j peanut oil suspension ( 0.5 rng)
per g of body weight ~
Fetuses at the 18th day of ge>tatiou
Day of Total Total '
mjecti- No. of No. of ~"r-, ) ~, (col x T°
on | mothe- . implan- suruved resorbed dead
rs tations •
_
survivors body weight
; malform- raeaa±^tand-
i ed < ard error '
_! No. and type?* of :
; ) , each anomalies j
8- 9
62 40 (01.5) !l'l' (33.5; i 0 ( 0; ; 5 (11'. 5) ' 1. 19±0 LI4 1 Dt, 4 Kn. 1 Pd
10
82 '49 C59.&) .« (40. 2 > 0(0) 5 (10.2) | l.C3±0.01S i 3 Dt, 2 Kn
1--13J 14 i 107 ]71 (ti'i.4- |35 (32 7) j i ( 0.0) i 3 ( 4.2) | 1.20±0. H13 1 Cp, 2 Dt. 1 Kn j
H-15 j 7 j 57 !.37 (iM.91) IS (31.«) ' 2 ( 3.5; ! 2 C :. I) ' 0. 9.j±0. Olu 1 Ct. 1 Dt '
* Cp: cleft palate, Ct: curvefi tail. Dt: deviation of toe or finger, Kn: malformation of knee
joint. Pd: polydactyly of tee.
X : °0 of total No. of implantations, ^ : °0 of survived.
127
-------
Table If. Effects of monoraethylaminoaiobenzene on the offspring of mice ^Intrapento-
neaJ injection of 0.004 or 0.006 ml 5% peanut oil solution ( 0.2 or 0.3 nig) per
g of body weight""
Day of
injecti-
on
8- 0
10-11
12-13
14--15
Total
No. of
mot he
7
12
19
7
Total i FetuseS at "
No. Of , (»f) x; (»,) x
implan-; survived ' resorbed
tat ions { i
1 f
61 i 40 (65.6) pi (34.4)
81 J36 (44.4) J45 (55.6)
121 145 (37.2) [76 (62. S)
54 \il (57.4) 23 (42.61
\e 18th das
(M x
dead
0
0
0
0
• of gestation
(°o) i. ( g)
survivors body weight
malform- mean^tand-
ed ard error
4 (10.0) i 1. 11±0. 023
1
4 (11.1) \ 1.03±0. 023
4 ( 8. 9) 1.06 ±0.013
1 ( 3.2) 1. OJ±0 019
No. and types* of '
each anomalies '
f
IDt. 1 Kn.lMd. 2St
lAd. ICp. IDt,
1HI. IPd
2An. IKn, IPd
!Pd
* Ad. adactyly of finger. An: malformation of ankle joint, Cp: clef palate, Dt: deviation
of toe or finger. HI: harelip, Kn: malformation of knee joint, Md: macrodnctyly of tee,
Pd: polydactyly of toe, St: short tail.
x : °,o of total No. of implantations, A : °«of survived.
Table EQ Effects of 3 t-fluoro-4-dimethylaminoazobenzen6 on the offspring of mice
£Intraperitoneal injection of 0.006ml 5 fj peanut oil suspension (O.Smg) per g of
body weight^
Day of; Total ' Total ,
injecti- No. of j No. of
Fetuses at the 18th day of gestation
_ I No. and types* of
g) i each anomalies
on
8- 9
10-11
12-13
14-15
mothe-
rs
9
10
9
9
implan j survived
tations ,
!
45 ,39 (86.7)
j
69 125 (36.2)
..
59 J31 (52.5)
57 |24_(42.1)
resorbed
6 (13.3)
44 (63.8)
27 (45.8)
33 (57.9)
dead
0 ( 0)
0 ( 0)
1 ( 1.7)
0 ( 0)
survivors body weight |
malform- inean±stand- '
ed ard error <
5 (12.8)" O.S2±0.u24 j lAn, IDt, 2E1, IPd
4 (16.0) 1. 15±0. 030 ! 2Dt. 1E1. 2Kn
3 ( 9.7) 0.87 ±0.034 j 3An, iCt, 1E1, 2Kn
i
1 ( 4. 2) 0.90+0.027 ! ICp
* An: malformation of ankle joint, Cp: cleft palate, Ct: curved tail, Dt: deviation of toe
or finger. El: malformation of elbow joint, Kn: malformation of knee joint, Pd: polyd-
actyly of toe.
X : fti of total No. of implantations. A : I'o of survived.
Table 51. Effects of 4'nuoro-4-dimethylaminoazobenzene on the offspring of mice "Intraperi-
toneal injection of 0.004 ml 5ae peanut oil suspension ( 0.2 .ng ) pt-r g oi tody
weight 3
Day of
injecti-
on
8- 9
Total
No. of
mothe-
rs
7
Total
No. of
implan-
tations
55
Fetuses at the
survived
41 (74.5)
18th day
Co) X' (°a) Xj
, resorbed , dead i
I ! !
!U (25.5) ! 0
( 0) '
of gestation j
survivors
malform-
ed
3 ( 7.3) |
i e ) •
body weight
mean ±stand-
ard error
1.07±0 OJ5 !
No. and types*
each anomalies
lAn.
2Kn, IKt
of
; 10-11 j 11
! 12-13 '
I
i 14-15
71 '28(39.4) 43 (60.6) j 0 ( 0) I 8(23.6): 1. 14±0. 023 2An, 4Dt, 1E1. 4Kn
I
11
85 38 (i.8.2) ;25 (29.4) ! 2 ( 2. 4) ! 2 ( 3. 4j 1.00±C. 0.0 ICt. IKn
i
44 40 (90.9} ' 4 ( 9.1) j 0 ( 01 0(0)
0.88 ±0.019
* An: malformation of ankle joint, Ct: curved tail, Dt: deviation of toe or f-'nger. El:
malformation of elbow joint, Kn: malformation of knee joint. Kt. kinky tail.
X .' "•> of total No of implantations, f_, : ', of survived.
128
-------
Table fit-Evaluation of the lethal effect of
peanut oil injection upon fetuses
by x:-test : control groups with-
and without injection were com-
pared with each other
Day of i „ „
injection
lo-n r.'-;;i 14-13
i :
i - ; - ;
< * : significant at 1 'a level,
* : significant at 5 °o level,
— : not significant.
TablefA Evaluation of the growth suppress-
ing effect of peanut oil injection u-
pon fetuses by F-te=t : control gro-
ups with and without injection were
compared with each other
j Day of ,
I injection
3-9
12-13
I
• " : significant at \ "0 level,
— : not significant.
Table S*\- Evaluation of the lethal effect of various derivatives upon fer-jses by xj-test com-
pared with the control group treated with the solvent
"treated" 3'-tnfluorom- moncethyla. ' 4'-methyl-l-d- mocomcthyl- ', 3'-fluoro-4-dt-' 4'-fluorc-l-ii-
v ' ethvI.-J-dime- mmoazoben- .' imethylan
;Dayof
.injection
thylammoaz- zene
thylamm ,'aminoazoben-. methylamm- . methylaramo-
! oazobenzene , zene ' oazobenzene azobenzene
8- 9
10-11
12-13
14-15
* ' : significant at 1 °a level, - : significant at 5 ",, level,
— : not significant, ( ~ ) : inverse effect at 5 a'a significant level.
Table fC Comparison of mortality between the fetuses treated by various derivatives on the
8th to 9th day
Derivatives
treated
a':irirtuoromet-,r_methyl__)_d
nyl-H-oime nyl gthylammoaz
Fetuses ^ - ene"~" ' ''?*™*
No. of resorbed
or dead
No. of survived
73 j 22
34 ! 40
J ; 107 j 62
__ „ — .
m- • monomethylam-
o- i inoazobenzene '
|
i 21
; -
i 61
4'-fluoro-4-dim- |
ethylaminoazob-' 2)
enzene
14
41
55
130
155
285
df = 3, x~= 36.73, P<0. 01
Table f4-Comparison of mortality between the fetuses treated by various derivatives on the
10th to 11th day
\ Derivat -
' ives tre-
ated
Fetuses
No. ot res-
orbed or
dead
No. of sur-
vived
2
: 3''tr'flr,T monoethyl-
1 -S- =T
, moazobenz- benzene
ene
71 ; 56
1
78 | 73
149 I 129
4'-methyl-
4-dimethyl-
aminoazob-
enzene
33
monometh-
ylaminoaio-
benzene
45
-59 36
82 81
3'-fluoro-4-
dimethyla-
minoazobe -
nzene
44
25
69
!
4'-fluoro-4- I
dimethyla- v
romoazobe , ""
nzene |
43 j 292
1
23 289
71 | 581
df=5, r= 15.13, P<0.01
129
-------
Table 5"7 Comparison of mortality between the fetuses treated by various derivatives on the
12th to 13th day
\ Derivatives '
^X^ treated
Fetuses — __ '
No. of resorbed \
or dead . i
No. of survived ;
2 j
3'-tnfluoro-4-dimet-
hylaminoazobenzene
57
57
monome th y 1-am mo-
azobenzene
76
45
114 121
3'-fluoro-4-dimethyl -
amino-azobenzene —
!
28 161
31 133
59 294
df=2, -^ = 5.44. p>0.05
Table SX Evaluation of the teratogenic effect of various derivatives upon fetuses by
compared with the control group treated with the solvent
(some cabes are calculated by exact method/
DenvatKeb-
"treated" ^'-tnrluorom-' monoethyla- ; 4'-methyl-!-d- monomethyl- 3'-rluoro-4-di- 4'-fluoro-4-di- |
i ethyl -J-dime- . mmoazoben- j imethylanin- ammoazofceri- : methylamm- methvlamm- ;
Day of ' v
injection
8- 9
10-11
12-13
14-15
tnyiammoaz- zene
obenzcne i
| _•
. . i «
|
1 _
(
i
oazoix-nztrne ztne oazoberuene oazobtnzene
i i
i . i
j '
— ! — — ! —
* =- : significant at 1 "o level,
" : significant at 5 "« level,
— : not significant.
Table fl- Comparison of teratogenicity between the fetuses treated by various derivatives
on the 10th to llth day
Denvat- 3'-tnftuoro
' ives tre-' methyl- 1-d
ated imethylam
inonzoben.z
Fetuses ' ene
• Malformed
j Normal
!
monoethyl-
" ammoazob-
enzene
15 i 5
63
78
j 63
i 73
4'-methyl- , monometh-
4-dimethyl ' ylaminoaz-
aminoazob- oberuene
enzene j i
5
44
49
4
32
36
riir.iethyla-
minoazobe
nzene
4
21
25
4'-fluoro-4-
dimethyla-
minoazolx;-
nzene
j 8
i 20
i 23
.
41
24S
289
5,
'~~ 10.56, P>0. 05
Table 4*- Evaluation of the growth suppressing effect of various derivatives upon fetuses by ,
F-test compared with the control group treated with the solvent
Derivatives
treate'd"! 3'-trifiuorom moncethyla- I 4'-meth>l-4- i monomethyl- i 3'-Huoro-4-di- 4'-fluoro-4-di-
i ethyl-4-dime- mmcazobenz dimethylami- : ammcazobe- j iiethylamino- oiethylamm-
| thylamintaz- ene
', noazohenzene , nzene
azobenzene i oazobenzene
8— 9
10-11
12-13
14-15
• *
• -
—
"
1
(-•) j
(• •) j c- •)
>.
(-
- • : significant at i "<, level. - : significant at 5 '<> level. - : not significsint,
( ' ) : inverse effect at 5 °0 significant level, ( • • ) : inverse effect at 1 °a significant level.
130
-------
Table fc'- Comparison of tiie growth suppressing effect between the malformed and normal
fetuses treated by various derivatives en the 10th to I3th day by F-test
Derivatives
-. treated
Day of , i
. injection X|
10-11
• 12-13
S'-lrifluorom -
ethyl-4-dime-
thylaminoaz-
obenzene
-
—
monoethyla-
mmoazoben-
zene
i 4'-methyl-4- i
: dimethylamin
1 oazobenzene ;
1 j
mono methyl -
ammoazoben-
zene
3'-fluoro-4-di-
methylamin-
oazobenzene
!
!(••)'
i ! ' !
l'-fluoro-4-d-
imethylamm- :
oazobenzene ,
~ i
1
— : not significant,
• ) : inverse effect at
1 *,, significant level.
Table tfc. Occurrence of malformed ribs in
the offspring of control mice tr-
eated with peanut oil
Liay 01
injection
No. of
mothers
No. of
fetuses
No. of fetuses
with mal-
formed ribs
8 — 9
8
47
0
10-11
2
11
0
12-13
2
10
0
14-15
5
26
0
. Tabl*>3- Occurrence of malformed ribs in
the offspring of mice treated wi-
th 4'-methyI-4-
-------
Table 4^- Occurrence of malformed ribs in
the offspring of mice treated with
4* -ftuoro-t-dimethylaininoazobenzetie
CO-2 ,wg,'g (body weight))
Day of
injection
No, of
mothers
No, of
fetuses
No, of fetuses
8-9
5
41
10-11
6
28
12—13
9
58
U--15
7
40
with maliotna-'
ed ribs ,
— : not significant.
Table fcl Classification of fetuses according
to No. of ossified sternebrae in co-
ntrol mice treated with peanut oii
\ Day of
% injection
N'o. of N
stained -
' sternebrae ' \
Q , ^
j
, 3-,
5 — 6
Total
8-9
0
0
.17
•4T
10-11
0
0
11
n
12-13
0
0
10
10
»-
0
0
2b
2S
Table t€. Classification of fetuaes according
to No. of ossified sternebrae in mice
treated with 4'-methyl-4~dimethyla-
minoazobenzene
[0. 3 mg/g (body weight)]
Total
40
49
: not significant.
\ Day of
\ injection
No. of ^
stained
8-9
sternebrae \ !
0 — 2
3-4
5-6
0
10—11
~
0
12-13
-
. 1 i 0
39
0
49
70
14—13
0
o
V
3,
Table kft Classification o< fetuses according
to No. of ossified sternebrae in
mk» treated with monomethyla-
uimoaaobeniewi
CO.2 or 0,3 mg/g (.body weight)
x Dav of
injection
No. of
stained
sternebrae x
8 — 9
10-11
0-2 i 1
0
3-4 5 j 1
.-.
Total
.
34 i 35
,
4<~/
36
1
12-13
0
1
44
45
i
14—13
0 i
0 j
1
31 i
3, I
* : : significant at 5 °a level,
— : not significant.
Table "W Classification of fetuses according
to No. of ossified sternebrae in
rsice treated with 3'-fluoro-4-dinj"
ethylamiaoazobenzene
CO. Smg.'g (body weight) ^
Table 71 Classification of fetuses according
to No. of ossified sternebrae in mice
treated with 4'lluoro-4-diuiethy!ami
noazcbenzene
0.1' m&'g (body weight)!
\ Day of
v injection
No. of .
stained -x
sternebrae X,
0-2
g ^
5-6
i
1
j Total
8 — 9
-4-
1
3
10—11
0
0
i
35 : 25
39
12—13
14-15
1 i •
0
0
I
29
24
25 | 31 1 24
x Itey of
injection
No. of • ! 8-9
stained j
stsrtiiitjrjic
10—11
12-13
0 — 2 i "I ! ~
i
3 — 4 [ 1
i
5 — C | 40
o
1
2T
0
_
0
58
Total j 41 j 28 | 58
14-15
"~ i
0 ,
3 i
»
40
significant at 10"o level,
: not significant.
: not signi Scant.
-------
Table 7L No. of fetuses with mislocated os-
sification center* of steraebrae in
control mice treated with peanut
oil
Table 7J. No, of fetuses with mislocated os-
sification centers of sternebrae in
mice treated with 4'-methyl-4-dim-
ethylamincczobenzene
Day of
injection
Mislocated
Normally
located
Total
8- 9 ' 10-11
1 i 0
46 i 11
47 j H
12—13 j 14—15 1
0
10
10
0 i
26 j
. 26 j
CO.Smg'g (body weight)]
Pa* ot s - a
injection !
Mislocated i ~
Normally
located
Total
3T
i
40
10—11
7
42
12—13
1
69
49 j 71
14—15
2
35
37
— : not significant.
Table 7^ No. of fetuses with mislocated
ossification centers of sternebrae
in mice treated with monometh-
ylaminoaiobenzene
^0,2 or 0. 3mg,'g (body weight)]
Table TSTNo, of fetuses with mislocated os-
sification centers of sternebrae in
mice treated with 3'-fluoro-4-dim
ethylammoazobenzene
| Bay of
i injection
; Mislocated
: Normally
• located
j Total
1 8 9
| 8 9
_$,.
! 6
i 34
!
] 40
! 10-11
i ,-,
1
i w
\ 26
t)
V
! 36 i 45
14-15
2
23
31
t «L £_. L&.V*!
1 Day of
! injection
j Mislocated
j Normally
"• located -
j Total
8 9
S
3! '
39 i
lo-n ;
1 i
24 i
]
25 1
12 -13
2
29
31
14 l.j
2
22
24
-s- : significant at 10S level,
— : not significant.
* : significant at 5 ?» level,
— : not significant.
Table <74- No. of fetuses with mislocated o:-
•sification centers of sternebrae in
. mice treated mth 4'-fluoro-4-dim-
ethylaminoazobemene
C0.2mg,'g (body weight) ~j
Day of
injection
Mislocated
Normally
located
~
Total
8-9
4
37
41
10-11
1
25
|
a i
12-13
2
56
» 1
14-15
1
3d
40
: not significant.
133
-------
Table 77 No. of fetuses with not ossified 13th rib in control mice treated wi'h peanut o.l
Day of injection
right (nor
left (1)
Staining
of the 13th rib
Not stained
Faintly stained
Stained
Total
r.
i
9
i
45
47
-9
1.
1
1
0
4G
47
10-11
r. 1.
0 Ol
o jo
0 0)
11 11
11 11
12-13
r. 1.
0 0
0 0
0 0
10 10
To 10
14-10
r. 1-
3| 4|
4 i4
il o!
• )•> oo
J6 W
Table Ti. N'o. of fetuses with not ossified 13th rib in mice treated with 4'-m*thyl-4-dimeth-
ylarmnoazobenzene
L 0.3 mg/g (.body weight,,]
-~-^ Day of injection
--- right (r} of ' 8 — 9
leftc.1) i
Staining i r' 1.
of the 13th rib '"--- i
Not stained 1 13 i** 13l*«
i 115 lib
Faintly stained j 2l 3J
Stained | 25 24
Total • 40 40
10—11
r. 1.
10i— 7| —
13 10
3] 3)
36 39
49 49
12
r.
6 —
9
3
62
71
-13
•I.
8
2
b3
71
14-15
r. 1.
11— 0|-
4 4
3 4)
33 33
37 37
• ' : significant at 1 °0 level,
: not significant.
Table "ft No, of fetuses with not ossified 13th rib in mice treated with rronometbylamino-
azobenzene
ro.2 or 0.3mg,'g (body weight)~)
-^^ Day of injection
' .. right (t) of 8 — 5
left 0) i
Staining of ; r. 1.
the 13th rib " • J
Not stained | 6'-1- 7 **
1 T n
Faintly stained i 1 4
Stained ; 33 .'9
Total • 40 40
10-11
r. 1.
J ~~" 4* 1 ~~
6 \3
3 1 !
30 33
36 36
12-13
r. 1.
4i— a i —
j 8 | 6
4l 3l
37 39
45 45
14-15 j
r. 1. i
11- ^1-'
I •? 1 i
i|- or.
29 20
31 31 i
• ' : significant at l ",, level, •*• : significant at 10?» level, — : noi signific-ant.
Table 1i(0, No. of fetuses with not ossified 13th rib in mice treated with 3' fluoro-4-dimethyl-
ammoazobenzene
C 0.3mg;'g (body weight;;]
" ^ Day ot injection .
nghi ( r; or i
lett(l.) !
' Staining of ! r.
! the 13th rib _ _[ '__\
( Not stained " 13
8-9
1.
' Faintly stained
i Stained
0
10-11
r. 1.
12-13
r. 1.
11-15
r. I.
36
35
39
39
2
0
.'3
Io
0
— 1
1
n
„.
i !
29 30
31 31
31 —
3
0
— : not significant.
-------
Table ftl» No. of fetuses with not ossified 13th rib in mice treated with 4'-fluott»-4-dimefhy[.
aminoazobenzene
C 0.2mg/g (body weight)^)
IX_ Bay of inaction j
i ~ right (r) or : 8
-- .left (1) :
i Staining of - „_ i r.
• (he 13th rib " -- '
. Not stained j 2 —
1 o
, Faintly stained 0>
i Stamed 39
, Total , 41
-9 10-11 12—13
1. r. 1. r. 1.
2 — 0 0) 01— 0
2 00 I 0
0 j 0 01 11 0
39 ! US 28 I 57 58 j
41 i ','S 28 (58 58 !
14-15
r. 1.
31— 2 —
J3 2 j
Of 0 }
37 38 '
40 40 j
-- : not significant.
Table n. Classification of fetuses according . Table S3 Clarification of fetuse, accordm-
to No, of oSS.fied caudal vertebrae {o :;0. of ^M esuiaal vertebrae°
m control mice treated with pea- in mice treated W]th 4..meth>.s.,.
dimethylarninoa2obenzene
\_Day of in- 1
""*- jection
Sinld - j «»"» , 1^"
caudal ^ 1
vertebrae N I
0—1 10 0 i
2-4* 5 3 |
5-7 42 ( 8 j
Total i 47 ! 11 I
j ! C 0. 5mg;g (body weight);
} : j ""- Day of m-
12-13 I 14-13 jection
N°-°f ' 8 9
, stamed . o — »
caudal )
0 j 0 vertebrae ^i
7 ; 8 o ._. j **
3 i 18 , ^ ^
10 i 3d . '" 4
5 — 7 I,'
Total j 40
j 10-11 j 12—13 J 14-15
•*• i (»^-) ! **
0 0 j 0 •
31 13 36
! 1" I 56 1 '
i 49 ; 71 j 37
* ' : significant at 1 "„ level,
,-*• '• significant at 10°j level,
(* *) : inverse effect at j <
significant level.
Table ft Classification of fetuses according
to No. of ossified caudal vertebrae
in mice treated with monomethyl-
amnoazobenzene
•"" 0.2 or 0.3 mg;g (body weight)'-
'"Day o' in- ;
• - : <>gnincant at ; ", level,
• : significant at 5 -,- level,
— : not significant.
Table fi"£ Classlikation of fetuses according
to No. of o>-ilie
-------
Table Vt, Clarification of fetu^s m
to No. of ossified caudal vertebrae
m mice treated with 4'-fluoro-4-
dimethylatmnoazobenene
'^ 0. 2 rng'tf ''body weight) '
Table ??' Classification of fetuses according
to No. of ossified metacurpal and
phalangeal bones in the hand or
• ossified met/itarsal and phalanges!
bones in the foot in control mice
Day of in- i i ' treated with peanut oil
Jetton i | ! i ' ' Day of in-
No. of
stam-d
cauci.-ii
vertt-hrae
0 !
j -4
5 - 7
Total
8-9
**
0
o-
11
•H
10-11 j 12-H ; 14-13 , : Tota, iection
, • 1 i No. of
! ' stained
— I i bones (
0 ; 0 3 | , - , u- 5
13 ' 39 ' 30 ! , K 6-11
1"> i U1 7 i I ~ 12-17
2S . o ' 4". ' ' 0-5
: significant at I -,; level, | _8 ! 6—11
— : not si£'iificant. j "*" 12—17
; Total
j 8-9
I
10 — 11
;
0 • U
: 12
i 3")
j i
0
11
0
! n i o
; 35
11
1 47 11
^
4
4
4
3
3
10
i
H-15!
1
0 j
5 !
•i >
0 ,
6 !
JO t
-•(» |
Table *•• Classification of fetuses according
to No. of ossified metacarpal and
phalangeal bones m the hand or
edified metatarsal and phalar.geal
bones m the foot in mice treated
with -1 -methyl-4-dimethylamino-
azobenene
' 0. Jmg/g (bydy weight }
TabltJ 89- Classification of fetuses tccording
to No. of ossified metacarpal and
phalangeal bone--; in the hand or
ossified nie'.atarsal and phalangeal
bones in the foot in mice treated
with raonomethvlaminoa.zobenze-
ne
jection
Total
N'o. of
stained
bones
0-5
id 6-11
! 12- -17
^ i
£ . 6-11
U— 17
Total
8-9
0
9
33
;
33
4J
1
1
1 10—11
i
! o
i 0
! 40
i
! 20
i 27
1
49
! 12-13
1
i
CKX
.
i 0
' 2
1 >
! /-** i
I I :
i 69 |
71 ,
I
14-13 j
!
i
;
2 !
64 !
21 i
i
***•
20 !
10 !
3" ,
v , Day of in-
" *• jection
Total ,
No. of -
stained
Dontrb
Q ^
C
K i 6-11
! r 17
! 0-3
I j 6-n
Total
8 — 9
*
tj
q
9
11
20 ,
L
40
10—11
r
1
13
22
*
8
9
19
3*i
12-13
(.**)
1
4''
12
32
-to
14—15
—
1
8
22
*
3
14
14
31
at 5 °; level, — : nor significant,
( • ) : inverse effect at 5 •> significant level,
( • • .: : inverse etYect a.\.\ '; significant level.
Table 90. Clas^ificr.tion of fetuses according
to No.of ossified matacarpal and
phalangeal bones in the hand or
ossified metatarsal and phalangeal
bones in the foot in mice treated
with 3'- fluoio-4-dimethylamino-
azobenzene
C 0.3 mg/g (body weight)
1 * : significant at [ "3 level, - : significant
at 5 °c level, - : no. significant, (**) : in-
verse effect nt 1 °Q significant level.
Table 7/« Classification of fetuses according
to N'o. of ossified metacarpal and
. phalangeal bones in the hand or
ossified metalarsal and phalangeal
bones in the foot in mice treated
with 4'-fluoro-1-dimethylaminoazo-
benzene
C 0.2 mg/g (body weight) 7
Day of in-
jection
Total 3 _
No. of
stair.ed i
bones \ i
_ ' 0-5 p
£ ' 6-11 ] 16
12- 17 1 1
+• *
• 0- 5 ; ,-
U ll
5 6--11 11
' 12 17 H
Total :v>
: bixiiiticunl 1
t
9 10- 11 ; 12-13
i
14-13
0 10
j 11 14
11 7
**
5 n
,
8
10
««)*•
10
'9 13 i 13
11 4 ' 1
-'./ , 31 ''I
t ",, level.
riot si«n;f.iant.
i Day of in- i i
1 jection i
i Tatal s „
j No, of 8 " " H
i stained
1 bones ' N
; _. 0- 5" i ~T
i £ : 4
\ ~ 6-11 ! 13
j ' 12-17 21
10- 11
9
i
12-13
14-13
!
t
Q
3
20
Z>, l 30
J ; ** i
i ^ ' °" " 10 3
i | ! 6 11 6
! '• I'.' - 1", 25
Total ' ii
3
'.;.;
19
Hi
';:',
:'» ' >, ' '.M
3
n
2.1
Jf
8
11
lf).
" 'ID
' • ; i pxnil irtnl nt| "i Irvrl, ' : sIKiillU'iint
' af :. "„ Irvi'l, ' : nl^riUiriinl ttt !(!•'„ ItVfl.
3''.
-------
Kelly et al (1964) acquired eight samples of commercial
Trypan Blue, including a pair especially prepared to contain extra
"red" contaminant. Half gram units of the as-received dye were made
up to 50 ml in 0.9% saline. Other 1/2 g units were Soxhlet extracted;
the thimble residue, mostly blue dye, was made up to 50 ml, and the
extractant, mainly red dye with very little blue, evaporated down and
made up to 50 ml in saline. No attempt was made to adiust the whole,
blue, or red solutions from the various suppliers to the same "concen-
tration." On each of days 8, 9, and 10 of gestation, rats were given
1 ml i.p. injections of one or the other of these three test solutions.
They were sacrificed on day 20. Resorption and malformation statistics
are given in Table 92. The malformed fetuses showed reduced body size,
edema, exencephaly, spina bifida, but rare caudal defects.
The teratogenic action of the whole dyes did not
correlate with actual dye content (53-82% blue, 3.6-13.7% red—the two
special lots, dyes 7 and 8, had 22 and 35% red).
Lloyd and Beck (1966) purified some commercial dyes and
one especially-prepared dye, all related to trypan blue: Afridol blue
(91%), Evans blue (100%), Niagara blue 2B (90%), and Niagara blue 4B
(91%). Rats were given s.c. injections of 1% aqueous solutions at 8.5
days of gestation, and then sacrificed at 20.5 days. Variations of
treatment involved sacrifice of Niagara blue 4B-treated rats at 11.5
and 14.5 days, and sacrificing Evans blue 7.5-day injected rats at 20.5
days.
It had been determined that both sexes of this rat
strain responded the same with respect to serum dye levels after
-------
injection. A number of male rats were given s.c. doses of the 1%
solutions; their serum dye levels were determined at intervals of 12
hours (each determination required a different rat as sacrifice was
involved).
TABLI-; 92.
Kn u is OF TRYIMN Bl.ut <>;* Ftiusts *
Dye
Whole dyes
1
2
3
4
>v5
6
7
8
Illuc fracliorn
1
2
3
4
5
6
i
J>
Ki-ii fraction
I
7
.1
-1
5
6
7
8
AH dyes
Controls
No.
litters
5
3
3
6
3
3
3
3
29
5
4
2
3
4
3
2
3
26
3
3
;t
6
3
3
2
3
26
81
16
No.
fetuses
38
25
29
31
29
33
29
28
242
35
29
16
22
30
13
11
30
180
23
.'7
32
10
2V
1 1
2l
.11
2,iV
66 S
1VO
Litter
sue (a\r.)
7.6
8.,-t
9.7
S.2
9.7
11.0
9.7
9.3
88
7.0
7..T
8.0
7.3
7.5
4.J
5.5
100
7.1
7 V
90
10 7
6 7
90
11.3
21.5
10.3
9 5
8.S
10X
AnoiiiiiliMis
individuals
No. %
12
8
11
17
3
1
1
3
56
5
3
0
1
0
•1
(I
0
13
1
0
1
1
0
2
2
1
g
77
3
- 31.6
(2.0
37.9
51 K
103
3.U
3 A
10.7
21 1
14 3
103
0.0
4 5
no
3' i !<
no
00
70
A i
II 0
i 1
J i
00
5 (l
I' ";
.', 2
3-1
I 1.0
1 8
Rcsorptions
No- %
12
6
2
19
4
0
0
4
47
9
4
1
1?
0
20
g
i
5f>
fr
0
<1
c;
o
0
0
0
17
120
2
24.0
19.4
6.5
37.0
12.1
00
00
12.5
lb.3
204
12.1
S.9
35.3
00
60.6
450
3.?
23.1
20.7
00
11.1
11,1
69
00
00
00
6.7
15.3
1.2
Resorption-abnormality statistics are given in Table 93.
Figures 5, 6, and 7 present maternal deaths as a function of dosage,
resorption-abnormality as a function of dosage, and serum level changes
138
-------
with time—all including trypan blue for comparison, results previously
reported. For comparison, in regards to Figure 5, the authors deter-
mined LD-50 values on males over a 12-day period, and found 179 mg/kg
for Niagara blue 4B and >400 mg/kg for Afridol blue.
Table 9i Teratogcnic response to four bisazo dyes injected
subcutdneously at 8-5 days of pregnancy
No. of mothers
Surviving Total Resorbed Abnormal Normal
Dye- Dose In- to implanta- , A-^-
(mg/kg) jected term tions No. %* No. %*
Niagara blue 2B
Niagara blue 4B
Afridol blue
Evans blue
50
100
150
200
50
75
100
150
25
50
100
150
200
300
50
100
150
200
11
13
14
7
13
12
13
11
10
14
8
11
10
9
7
8
8
10
11
12
12
5
13
11
11
5
10
14
8
11
9
8
7
8
7
7
97
114
128
53
122
109
114
50
123
134
77
H3
80
86
70
72
70
74
7
33
46
53
31
39
64
50
22
32
16
57
45
71
13
31
66
58
9-0
28-7
407
100
33-5
36-4
56-6
100
17-2
26-0
21-8
50-3
58-7
81-7
16-1
\
95-2
798
1
4
32
0
3
0
0
0
9
11
13
21
10
7
1
->
4
2
0-9
3-5
22-2
0
2-4
0
0
0
7-6
7-7
16-3
17-2
13-2
10-6
1-1
2-5
48
2-4
89
77
50
0
88
70
JO
0
92
91
48
35
25
8
5<)
W
0
14
90-
67-
1
8
37-1
0
64
63
43-
0
75-
66
61-
32'
2K
7
82-
W
0
17
•1
•6
4
2
•j
9
5
1
•7
6
1
9
* Percentages represent the arithmetic means of the percentage within each individual
litter; this enables the standard error of the mean resorptions and malformations for each
dose to be calculated and shown in Fig.4.
139
-------
so 4
I 30 ^
o
T3
£ 20 H
10-
• Niagara blue 4B
Evans biue
?
Niagara blue 28
Afridol blue
Tryp.n tlue
i t 1
SO 100 153 200 250 300
Dose (mg/kg)
Fig. 5. Marer.ial mortality within 12 days of administration of various bisazo dye
Figures 5-7 reprinted with permission from J. Embrol. Exptl. Morphol.
16:29-39 (1966). Copyright by Cambridge University Press,
-------
100-i
80-
60-
40-
20-
100-
80-
60-
40-
20 -
100-
80 -
60 -
40 -
20 -
100-
80-
40-
20-
100-
80-
60-
40-
20-
Trypan b!ut» |
/'
i-'Y
f'{''L
t^H^.
50 100 150 200
Niagara blue 2B //
/
f
t
t
T ---"f
l:'^^]^\_
i i i i
50 100 1^50 7.00
Niagara blue 48 f.'
y'
\~~~Y'
*•
1 - -r—
1 1 I!
50 100 150 2CO
Afridol blue
...-I- [-
|— .. Y'' T
f j — • — ~~r~ r~~~ 1 —
1 | t I
50 100 150 200
Evans blue /I--.
/ "i
.-I'''
Y"
, , f , , —
50 100 150 200
250 300
I'M 300
i I
250 300
-4
T
1
I i
250 300
1 1
250 300
Dose (mg/kg)
Fig. fe- Dosage-response.curves for the turutogcnic aclisity of five
bisazo dyes. , Resorptions; , abnormalities.
Trypan blue (174)
Fig. 7 Serum levels following injection of 50 mg/kg of various dyes
(figures in parcnthesi1: are the average levels over the first 24 h).
it. I
-------
The reason for giving an Evans blue injection at day
7.5 was the very slow release into the blood stream from the injection
site, and a high enough dose to get results comparable with the other
dyes would be too toxic to the mother. Thus, injections of 100 or 150
mg/kg at day 7.5 produced these changes from the same doses at day 8.5:
at 100 mg/kg % resorptions dropped to 30, % abnormalities rose to 12.7;
at 150 mg/kg % abnormalities dropped to 0.
The results of killing the mothers at 11.5 and 14.5 days
after a 100 mg/kg dose of blue 4B at 8.5 days indicated most of the
resorptions occurred by 11.5 days, presumably from toxicity rather
than secondary consequences of malformation.
Beaudoin and Pickering (1966) synthesized 16 dyes related
in some fashion to trypan blue and gave them as 140 mg/kg i.p. injec-
tions to 8-day pregnant rats, which were sacrificed on the 20th day.
Autopsy samples of the maternal macrophage system, kidneys, placenta,
and yolk sac were examined for the presence of the injected dye.
Table 94 presents dose-comparable literature results on
some highly relevant compounds. Table 95 presents the five compounds
most closely related to trypan blue structurally, and results. Of the
remaining 11 dyes, none was shown to be a teratogen, nor was there any
found in the tissues examined—six of these dyes consisted of simulations
of the compounds in Table 94 cleaved at the biphenyl linkage. Table
96 presents the tissue distribution of the compounds whose structures
were given. The authors disclaimed Compound 1 as a teratogen, and
were reluctant to so label Compound 8, pending further study.
1U2
-------
TABLE - - (
EVANS BLUE
No SO,
HO NH,
J = N
CH,
CH,
NaSO,
NIAGARA BLUE 48
,N OH HO MH,
N = N-—
-------
TABLE f 5".
Effects of synthesized disazo compounds on rat gesl.ition
Number N|t!fJitrnof Embryos Survivors
of mother, "" resorted '
COMPOUND 6
HO OH . HO OH
M.II-/* W VN«N
~Vyv_<^
CH, CH,
7 69 22 0
COMPOUND I
OH HO
NoSO,
COMPOUND 6
,N OH HO
...H^XQ....
.No
NoSOj v V" So3Na
6 48
4 2
8 76 14 3
COMPOUND 5
H»N OH HO NH,
' .-f>Q". '
NflSO,
COMPOUNO 10
OH HO
OCH,
OCH-
NoSO
5 53
6 60
0 0
7 0
- TABtE 96.
distributiori of disazo dues and selected synthetic coi,ipound$
Maternal macrophage
cells of liver, spleen,
lymph node and lung
Yolk sac
epithelium
Trypan blue
Niagara blue 2B
Evans blue
Niagara blue 4B
Niagara sky blue 6B
Compound 6
Compound 1
Compound 8
Compound 5
Compound 10
+4-
+4+
Ikk
-------
Pizzarello and Ford, Jr. (1968) dissolved 6 mg of 4-dimethyl-
aminoazobenzene in 0.1 ml of polyethylene glycol or PEG/ethanol (9/1) and
injected it through the shell and air space into the yolk of 2-day old
chicken eggs. From the PEG injection all of the surviving chicks had
shortened leg bones, and half had deformed feathers. From the PEG/
ethanol injection most had shortened bones, and 60-70% had deformed
feathers.
Stein et al (1969) injected 10 ug of Janus green B into the
amniotic fluid of incubated eggs at the 29 Hamburger-Hamilton stage.
All of the survivors exhibited syndactylism.
5. carcinogenicity
Reports of tumors resulting from repetitive injections at the
same site have not been included unless the tumors appeared other than
at the injection site. Reports dealing with anti-cancer testing of azo
compounds, and any metabolic-physiologic information contained therein
have been incorporated into this carcinogenic reports section.
Seligman et al (1952) found that growth of sarcoma 37 in mice
and Walker carcinoma in rats was inhibited by l-methyl-2-(phenylazo)naph-
thalene and l,4-dimethyl-2-(phenylazo)naphthalene. Sarcoma 37, only,
was inhibited by 3-phenylazophenanthrene, 2,2',5,5'- and 3,4,4',5-
tetramethoxyazobenzene. Walker carcinoma, only, was inhibited by
3-phenylazoacenaphthene, 3,3'-dimethylazobenzene, and 3,3',4,4',5,5'-
hexamethoxyazobenzene.
Simpson (1952) gave rats s.c. injections of 10 mg of Trypan
Blue every two weeks for 14-16 weeks in some, much longer in others.
Of those given the continuous dose and surviving for 151-250 days, 12 of
-------
21 had hepatic retlculum cell sarcomas; 6 of 9 surviving 251-350 days
had this tumor. Of those given 14 doses in 182 days and surviving 210-250
days, 3 of 5 had tumors. The author was unable to demonstrate the
t >:ansplantabillty of the tumors in 24 attempts, but may not have allowed
sufficient observation time. He was aware of the impure nature of the
commercial dye and was not at all certain that the tryp&a blue component
was responsible for the tumors, in part or in whole.
Miller et al (1953) fed rats 4-dimethylaminoazobenzene (DAB)
with 1, 2, or 3 fluoro groups in the non-amino benzene ring for comparison
oT carcinogenicity with DAB itself. The results, in Table 97, were
interpreted as meaning that the carcinogenicity of DAB did not involve
£.••>* of the o-, m-, or p-positions of the non-amino ring. Also tested,
ai.d also found to be more carcinogenic than DAB, was 2-fluoro-DAB (Series
II. Group 6).
TABLE 9?,
THK CARCINOGF.NICJTIKS OK VARIOUS FLUORO DERIVATIVES OF ^DIMETHYLAMINOAZOBBNZENE
Btoim
1
11
HI
IV
V
•No.
Ggour
1
2
3
4
5
6
7
8
9
10
11
12
IS
14
16
• niuuitf
COHFOUKD rro
DAB
2'-Fluoro-DAB
3'- " «
4'. «
DAB
2-Fluoro-DAB
«',4'-Diiiuoro-DAB
DAB
2',S'-Difluoro-DAB
a'.s'- " «
DAB
a',4',(i'-Trifluoro-DAB
DAB
" -f- wxlimn
fliiorouwlnte
Sodium
fluoruuretute
with tumom/ttuiulier of ftDiuiitb nib
T»MB
COMHHJND
PlB O»T WAS Ti.0
JK J>IET (MO.)
0.054 ' S
O.OS9
«
"
0.054
0.050
0.063
0.054 4
0.063 S
H «
0.054f 3
-0.045
O.OCGf *
-o.«wo
(UK! 4
O.CMIf
O.OOi
0.002 10
v &t end of 4yr frrdil^{.
IHC,O«CE or
uv«a tu»< omi*
(MO.)
S 4
S/IS
4/13
8/14
18/25
Z/16
3/1 S
10/16
8/16
9/16
9/14
I/IS
8/15
8/1S
• 8/1S
Reprinted wi
Research 13:
9 S
7/15
8/1S
lii/14
2-4/45
3/16
13/15
1C/18
11/16
16/16
H/14
6/15
13/15
7/15
7/15
0/1(1
(ttml nt
10 nHW.)
th permission
93-97 (1953).
Cancer Research Inc., and
Association
(xSOBB CIHKiiOSia
AT EfW OF
rEOTINO C-OMMJUNB
none-mild
mild
moderate
11
none-mild
niild-iirioderate
moderate-severe
none-mild
moderate
a
none- mild
moderate-severe
notuMiiild
M U
none
from Cancer
Copyright by
the American
for Cancer Research.
Ih6
-------
Groups 11 and 12 received the high "% in diet" only for the
first week, that level of the trifluoro-DAB proving too toxic, and it
being desirable to treat the DAB controls the same as the test group so
far as molar amount of the dye given. Series V was an attempt to determine
the carcinogenicity of a possible metabolite of the fluoro groups,
fluoroacetate, but it was too toxic to be given at the maximum potential
level; it didn't show any carcinogenicity at the maximum level tolerable
to the rats.
Nelson and Woodard (1953) fed dogs o-aminoazotoluene (AAT) or
4-dimethylaminoazotoluene (DAB). The dose of 20 mg/kg/day of AAT killed
all the animals within eight weeks from hepatic damage (no tumors). The
same dose of DAB killed 8/10 dogs in 16 months (no tumon) , the remaining
two having tumors. At 5 mg/kg/day AAT produced no tumors in four months
in one dog, and tumors in four dogs in 30-62 months. The same dose of
DAB produced no tumors in six dogs in 63 months. Only AAT caused hepatic
and gall-bladder tumors, but both caused urinary-bladder tumors.
SchmShl (1954) fed rats 5-10 mg/day of 2-hydroxy-4-dimethyl-
aminoazobenzene until they had received 2.5 g. Weight gain was normal
and no tumors developed. The livers had a normal appearance.
Brown et al (1954) prepared some analogs of 4-dimethylamino-
azobenzene (DAB) in which the non-amino benzene ring had been replaced
by a pyridine, pyridine-N-oxide, or thiazole ring and tested them against
DAB in rats at 0.06% of their low-protein, low-riboflavin diet. Table
98 contains the tumor incidence, survivability, hepatic histology,
and 3'-methyl DAB comparison data. The latter was from a follow-up study
on P04; because of the latter's toxicity, one day each week for the first
-------
Reprinted with permission from Cancer
Research 14:22-24 (1954). Copyright by
Cancer Research Inc., and the American
Association for Cancer Research.
. TABLE 98.
TUMOU INCIDENCE* AMONG THE VARIOUS GROUPS OF RATS
RECEIVING HETEUOCYCLIC ANALOGS or DAB
Compound
i-a'/o-jMlimelhylnnilinc
t hylan
line
lammoazobenzcnc
•! liylnnilinc
:i hyliuulinc
Code
r-2
T-2
DAB
4 mo.
0/2
0/2
0/2
0/2
0/2
fi/o
1/8
0 mo.
0/2
0/2
2/2
0/2
0/2
8 mo.
0/2
0/2
4/4
0/2
0/2
10 mo. IS mo.
0/2
0/2
no
0/2
0/2
0/2
0/2
survivors
0/2
0/2
no survivors
2/3
2/2
2/2
ro. sur-
vivors
* Tumor iucidcnfv tj numlxr of Mts \vitli hrpiUic tumorfl/ntimlKT of rats sacrificed.
SUMMAUY OF HlS'KIUKJJCAI, DATA OllTAIM'.l) FROM Till'. LlVKItS OF If ATS
JtlX'CIVIiNG IIKTKIK (CYCLIC ANALOGS OF DAB
Code 4 ninnlU
I'-i Difl'uic. fatly changes to normal
l'-3 Moder.ile fatty efiun^es, less than
l'-l Slight fatty changes
T-2 Marked fatty changes
1'02 Normal
I'0-l Liver cut ircly replaced by papillary-
tj pc tumor, fibrous tissue reac-
tion, acute mfluimnatoiy necrosis
D.VB One animal \utii niuKiple tumor
nodules, fibrous tissue reaction,
inflammation, fatty changes; two
nuiinah, livers normal
Control Xonnal
0 wontlis
Moderntc fatty changes
Normal
Nodular tumors; two kinds of noo-
pl.'isin-liepatoinii and papillary
adcnocartinoma arising from bile
duels
Normal
Moderate fatty changes
No survivors
Two nnimtils with tumor masses of
liver cell type surrounded by
sliplit fatty changes; one animal
liver normal
Normal
R months
Moderate fatty rlinn^c.s
inc(!ulaiily of lobulnr
p:ittorn, some large or
double nuclei
nodules of necrotic tu-
mor of liver cell type
Normal
Normal
No survivois
Normal
RESPONSES OF RATS RECEIVING POl AND S'-METinr-DAB
Survival (•( months)
Avenge body weight of survivors
Wci;;lil chanjje (3 months)
Liver weiyht as per cent of body
weight
Food consumption (gin/day)
Turner formation (4 months)
PO-J
2/6
H5 gm.
— 19 gm.
15 per cent
4/5 hud massive tumor formation;
1/5 had nodulation and small tu-
mors
S'-Me-DAU
4/3
230 gin.
32 um.
9 per cent
12
3/5 had extensive nodulation to
definite tumors; 1/5 had slight
nodulation; 1/5 normal
six weeks only the base diet was given (also to the 3'-methyl DAB control)
Sugiura et al (1954) compared the hepatocarcinogenicity of
some compounds similar to DAB, in rats; four of these were new, the 4th,
7th, 9th, and 10th compounds in Table 99. Dye intake was initially
about 6 mg/day; this fell to 3 mg/day in those rats with liver damage
-------
TABLE ft, -Incidence of hepatic lumort in rats fed various aio compound*
Compound fed
N.N-Dimethyl-p-aminoazo-
benzene
N-Methyl-p-aminoazoben-
N-Ethyl-p-aminoazobcn-
1CI1O , .
N-MoUiyl-l'-rnethyl-p-amJ-
N-Mcthyl-4'-othyl-p-ami-
noazobcnzonc
Formula
CII,
>«3SSS=rv jCSSSSSBSV ./
CH,
CH,
_ __. ^,.,,J. /
O~N==N~v_I/~N\
C,H,
"Jm"-*r \
H
CH,
H,C—^^^— N«=N—^^^— N
II
CH,
" ™" >-_
H
No. of
ani-
mals
15
14
15
15
15
Percent
in diet
*"
0.060
0,050
0.000
0. 000
0.004
No. of
days fed
75-250
132-250
148-250
100-250
104-223
j Inci-
Livcr findings at autopsy*
~~
0
0
15
8
0
±
•
1
1
0
4
0
+
3
5
0
3
7
+ +
5
3
0
0
7
+ + +
6
5
0
o
1
don co
of liver
cancer
(per-
cent)
93
93
0
20
100
-------
TABUS 99. Continual
N-Methyl-2'-methyl-p-ami-
N-Methyl-2'-ethyl-p-ami-
.
N-methyl-3'-methyl-p-ami-
N,N-Dimcthy]-3'-methyl-
4'-hydroxy-p-aminoazo-
3'-(4-DimcthylaTninophen-
9H' CH,
H
CjHj CH,
," •— ^ X -* -'-— -•», f
/ \_N=N— < /~N
H
9Hj CH,
H
CH, CHj
HO— ^ tf~~ N'=N— ^ ^ — N
^ y ^ _y \
CH,
CII,
^ ^ — N==N—C /^ — N
\V__X N__J' \
CH,
15
15
15
15
15
0.060
0.064
0.060
0.068
0.060
98-250
74-250
141-225
107-250
100-2oO
10
15
0
15
4
1
0
0
0
3
3
0
6
0
2
0
0
6
0
4
1
0
3
0
2
27
0
100
0
33
•—indicates smooth, practically normai iivcr; a: imiiwics noiiu'ar cirrhosis with sdcnorr.atoas byp«rp!as!a; J- ^
elve liver caneiT without metaslasla; +++ Inillcutca cxtcnalvo liver cancor with mctcstnsls.
rtl?tlnct areas of cbolnnKlomn or hoixttoma; ++ Indlcutos citon-
-------
and tumors who ate less food.
Brown et al (1954, pp. 715-717) followed up their earlier 1954
publication (see above) dealing with the carcinogenicity of pyridine-
and pyridine-N-oxide azodimethylanilines. Their results are given in
Tables 100 and 101.
TABLE 100.
TUMOR INCIDENCES OF RATS VKD VARIOUS PTOIDINR ANALOGS OF DAB
4-Mct hylpyridine-2-nzo-jMlimcthjfl-
nniliiic
COM:
4-Me-P*
IN IHKT
(prr cent)
0.06
1 BO.
ma. 3 mo.
auiUue
4-]Uclliylpyritiine-]-oxidc-2-azo-}»" 4-Me-PO2 *
diiiH'tbykniUne
6-W<-ll]yljjyritiii)c-l-oxi(le-2-uzo-j>- C-Me-POZ "
dinirfiiylani'ine
2-Mctliylpyridinc-l-azo-p-dimethyU 2-Me-P4 " _ 0/2
aniline /
Pyridine-l-oxidc-3-azo-p-dirnetbyl- PO3 * / . 0/2
aniline
MUethylpyridine-l-oxide-i-azo-p- 2-Me-PO4 *
dirnetliylanilinet
3-iUeUiylpyridine-l-oxide-4-azo-j>- 3-Me-F04 * 0/1 0/2
tlinictliylatuline
jj-Uitncthylaiiiiaoiizo bcniene DAB " 0/2
2-McJh.v1pyridinc-l-oxido-4-uw>-p- a-Mc-l*04 0.02 0/8J 0/4 1
diniolhylaniliiie
2,0-I)ini<:tliy]pyriiM>Kr when numbrn in porrnllirw* urr rfuj-a /*
1/3
5 mo. fl mo.
0/2
0/2
0/2
0/2
2/S 2/2
0/2 3/3
no survivors
2/2 2/2
2/3
8 mo,
0/2
0/2
0/2
0/2
2/2
10 «o,
o/i
0/4
0/2
0/4
«/2
TABLE 101.
KS «»• RATS UKCKIVIXG 2-MB-TO4
ASI> »I'-MKTHYI/-])AB*
Survival nt 4 raontlis
Av. IKK!}- weight of survivor!
Av. Kriclil change nt 4
months
Av. liver weight as per cent
of body weight.
Tumor formn t ion n t 4
months
*-Mc-PO4
7/10
lf>7 gru.
0 8 gm.
27. 6 per cent .
7/10 lincl mnssive
tumor fornm-
tion; ;i/10 died
wiiliin 2 weeks
of .sliirt
m'-Me-DAU
10/10
200 gin.
152 Km.
& o por cent
5/10 jslipht
necrosis;
5/10 nor-
mal
*T!ic rurrm*wni .il'lttc 0.c-. an' dsjs nn l»n*nl did witb nn (jruuo^n. H»(H)-(>«tl)*I-(^)»lI-(ij.
Reprinted with permission from Cancer
Research 14:715-17 (1954). Copyright by
Cancer Research Inc., and the American
Association for Cancer Research.
-------
Badger et al (1954) tested a variety of azo compounds for
carcinogenicity in rats fed a low protein, low riboflavin diet in
amounts molar-equivalent to 0.06% for DAB. For DAB, 7/7 and 8/8 survivors
had tumors, for 4'-methoxy DAB, 4/10 survivors had tumors, and for the
following compounds there were no tumors (number in parentheses is %
surviving): 2,2'-azonaphthalene (90), 1-phenylazonaphthalene (50),
2-phenylazonaphthalene (60), azobis(4-dimethylamino)benzene (80),
4-methoxyazobenzene (70), and azobis(4-methoxy)benzene (80).
Bonser et al (1954) gave mice s.c. injections of 3 mg of
l-(2-tolylazo)-2-naphthol (Oil Orange TX) twice a week for 50 weeks.
Intestinal tumors appeared at 62 weeks.
Miller et al (1957) prepared a variety of DAB-related compounds
ind fed them to rats to determine the hepatocarcinogenicity against DAB
as a control. The results are in Tables 102, 103, and 104. These tables
also include the results of studies on possible metabolites and rearrange-
ment products. The authors concluded that the 2- position of the amino-
ring of DAB must not be substituted in order to retain hepatocarcinogeni-
city. In Table 105 is a listing of relative carcinogenicities of
substituted DAB's.
152
-------
TABLK l«t.
THE CAnciNOGKNictTira OF CERTAIN- FLUORO AND METHYL DEKIVATIVKS OF VDIMETUYLAMINOAZODBSZESE
M
W
TIME
COM-
Scmu Giuror
I
II
III
n*
*
1
e
3
4
$
6
7
8
9
10
11
12
IS
Number of rats
•*
COMTOOKB FBO
(1.40 millimoks/kg diet No. I)
C*Jw:Ni"^N(CMj>g
4-I)unctliylamino«obenzene(DAI5)
{"^NsN^jNOyz
S-Fluoro-DAB
O-NCP^
S-MeLhyl-DAB
P^NtONDI^
S',4'-DifIuoro-DAB
vb^Oott
S',4'-Dimctliyl-DAB
DAB
(T
Of^O-wyz
2,6-DiHuoro-DAB
DAB
O*fe"«%
8-Me%l-MABt
F F
s^N=NC)NChy2
t,6,3',fi' -Tet ra Huoro-D AB
'CW>«tt
*,6,2',4'.6'-PentHfluoro-DAB
DAB
r r
^Vl^NQuCH^
^~r F
2,fi,2',5'-Tctranuoro-DAB
with tumors/number of rats alive »l
rooxn
wn ru
(mo.)
4
8
8
3
e
4
7
4
g
6
4
4
6
Ko. or IKCH»K*CE or LIVER TUMORS*
> in TO (mo.)
SMnrED 5 448 7 > 10 11
16 4/18 14/16
IS 2/13 10/1S
11 0/11 , 2/llf
•
15 12/13 13/13 .j
12 0/12 2/12
16 6/15 14/15
14 0/14 0/14
16 3/18 11/16
16 0/16 1/16
12 0/12 0/12
6 0/6 0/6
16 5/18 13/18
10 0/10 «/10
Guana ciKuaotn
AT Jt-f 0 OF fCKI*-
1KQ OT COJIPOCM*
Mild-moderate
•'
Mild
None
Moderate-sever*
None
Mild-moderate
None
Mild-moderate
None
None
None
Mild-moderate
Mild
m
end of dye feeding. f Benign hepatomas. J MAB «» 4-monoroetoylammoazobenzene.
Tables 102-104 reprinted with permission
Wl/*ll"i4" k» t * ^ *« » « ** u r*_— _«... I -r
Cancer Research 17:387-98 no«m raPy_
-------
Sum
V
14
15
TABLE 101.
Tas CARCIXOGEMIGITIES OF VARIOUS REARRANGEMENT PRODUCTS AJ*D OTHER DERIVATIVES OF 4-DiMETim.AMiNOAZOBExzEXE
(2.87 nillimolei/kg diet No. I)
DAB
TIKE
COMPOBXD
WA» rto
(BO.)
4
8
No. or
•in
•TAtTED
14
IS
IXCIDENCI or uvm
4
4/14
«
12/14
10
11
0/13
11
GKX9CIU8OU1
AT E3D Or WXt&-
on» or
MUd-moder»U
None
VI
VII
IX
IT
2-Acetylainino-5-dimetliyl»inino-
diphenylainine
Benzo(c)cinnoline
11
0/11
0/6
None
None
18
19
20
81
22
23
24
!5
26
27
f*ct
S-Hydroxy-DAB
DAB
Nrl
2-Nltro-5-dimethylamino-
diphenylanrune
ON=NOr*'ia%^ Q"
DAB mcthoehloride
S-Hydroxy-DAB
(cf. group 17)
DAB
• vC^ N 'C3M°V2
4-Atnino-4'-dilnetliylamino*
dlphenylamine
n^/~y N =^y*>^ s%
4-Imino-4'-dimethj'laminodiphenyJ-
im'me sulfate
DAB
(^N^NCMjk
N02
S,4"Dinitroclimethy!aniline
DAB
&
•tktJ* ^MnwL,!-
4
8
8
8
4
8
8
4
If'
4
f
ie
15
16
6
15
15
15
It
10
15
R
uj
15/16 IS/18 Severe
0/18 None
0/14 None
0/6 None
12/15 '4/15 Moderate
0/14 None
0/15 None
4
e/12 12/12 Moderate
0/12 * None
10/15 12/15 Moderate
0/6 No DC
-------
TABLE 104-
TH8 CARC1NOGENICIT1ES OF VAIUOUS DERIVATIVES OF 4-DJMETmfLASIIXO.V7OBENZE.VE
Snuu Giour
XII— Canl. 37
XIII 33
S3
M 40
xn.
\j\
41
XIV 4?
43
44
XV 43
46
CovrouxD(i) JED
^2NQNiNCXHfc
4,4'-Bis(dimethylamina)-«zoben-
DAB
Cp0*fe
2'-Methoxy-l>AB
S'-Methoxy-DAB
Oy<>=!<>«>y2
4'-Metliovy-DAB
DAB
3'-MeUnI-]>AU
S'-Mcthovy-DAB
DAB
ovwiQ^'O*^
Mn.t4MOLE«/
IO DIET*
2.67
2.40**
2.40**
2.40**
2.40**
t.OO
2.00
2 00
2.14ft
2.14ft
Tim
fOM-
jotmn
WAI rr-D
(mo.)
15
4
6
3
6
S
3
3
3
3
No, or
KATB
• T.MU.
16
18
16
18
16
1G
16
16
14
14
INCIDENCE o
S 4
1/16 7/18
•o/>«
8/9 9/9
0/18
0/16
6/16
8/16
1/14 5/14 ^
6/13 12/13
(
e
10/16
5/16
6/16
4/16
16/10
IC/K>
4'Etiivl-D.\B
(MO.)
8/16
12/1C
1?
0/13
G&OflB CSMHZ1O81II
41 i»n or
rE£i>[KO or
None
Moderate
Mild
Severe
MUd
Noce-mild
Moderate
Moderate
Mild
Severe
jj C^r,>i"jr.«j may be poorly absorbed, since it was readily detected in iJ e feces. ffFed 2.67 mtllimoles of dye/kg diet for C weeks and then level of dye lUteil for re-
** Jed i.-" mi;i:'r-..V.c> of -lye "kg diet for 2 weeks, then on dye-free diet for 0 dsys and maining time because of toxicity of 4'-ct{lyl djc,
returned to level of dye listed above for remaining time because of toxicity of S'-mclhoxy
-------
TABLE 105".
TtfE CARCINOGENICfTfES OF VARIOUS RING-SUBSTITUTED DERIVATIVES OF
4-DlMETItYIAMINOAZOUENZCNE*
2 3
PCWTIOM
4'
3'
2'
2
3
2', 4'
2', 5'
3', 4'
3', 5'
2.6
2'. 4', 6'
2, 6, 3'. 5'
2, 5, 2', 5'
2. 6. 2', 4', 6'
F-
10-12
10-12
7
> 10
4
>10
>io
>10
>io
0
>10
0
4
0
CHi-
-------
4'-sulfaminophenylazo)-4-hexylresorcinol (the last three had no effec-
tiveness) . The cell treatment was temperature and azo concentration
dependent.
Rlittner and Brunner (1959) were unable to induce tumors in
rats by fortnightly i.p. injection of 1 ml of a 2% solution of Trypan
Blue (18-19 injections) or Evans Blue (12 injections). The animals
were observed for 7-9 months after injections ceased, then sacrificed.
Mulay and O'Gara (1959) fed male and female rats 4-dimethyl-
aminoazobenzene (DAB), 4'-dimethylaminophenylazo-l-napbthalene (DAN), or
4'-dimethylaminophenylazo-2-naphthalene (DA-2-N) with the results in
Table 106. The DAB and protein 8 treatment was an 8% rather than the
usual 12% protein diet. Except for DA-2-N the sex difference in tumor
development was striking. Average time for tumor development from DAN
was 20% longer in the females. This figure was considerably higher from
DA-2-N treatment.
Table 106
[uciilcMcc of I.ivtrr Tumors in O^lioiiir-Mi-inlfl J'fits Y«~[ Oir(-ii.fi£,'''ii'- l>yr-,.
Treatment
, — Carcinogen — v
% Diiys fed
BAN
DA-2-N
DAB
DAB
and protein
.075 300
.15
.3
.6
.075 230
.00 280
.01)
8
Tumor- i
Induction , >'*; Ineiilciiff r/r
270
80
150
••
4?,/:>7 -.-,
2/4 a»
39/40 M
56/oG 8."*
:,,.ju;,
-------
month, 65% developed tumors. Incidence of tumors in second genera-
tion mice was nearly four times higher than that seen in controls.
Brown and Hamdan (1961) fed a variety of 4'-alkyl substituted
4-dimethylaminoazobenzenes (DAB) to rats at 0.06% of the low-protein,
low-riboflavin diet and examined them at two-month intervals for hepatic
tumors. DAB gave a 90% incidence at six months. The n-Bu DAB gave a 43%
incidence in 12 months (0% at six), the t-Bu DAB 33% at 12 months (0% at
six), the EtDAB and i-PrDAB 100% at four months (toxic), the n-PrDAB
78% at six months, and MeDAB, iso-BuDAB, and sec-BuDAB 0% at 10, 12 and
12 months; the phenyl DAB gave 0% at six months. At the 0.03% dietary
level the iso-PrDAB and EtDAB were 50% and 267% more active than 0.03%
DAB at six months.
Arcos and Griffith (1961) found that a seven-month feeding
of 0.04% 2-methylDAB or 0.02% 3'-methyl DAB to rats gave 0/22 and 1/23
incidences of hepatic tumors, respectively, but a combination of these
two dietary levels gave a 5/20 incidence at seven months. While 0.035%
of 3'-methyl DAB gave incidences of 14/24 at four months and 24/24 at
six months, adding 0.035% of 2-methyl DAB to this diet gave incidences
of 10/24 at four and 21/23 at six months. Feeding rats 0.06% 2-methyl
DAB for three months prior to five months of 0.054% DAB had no effect on
tumor incidence.
Takayama (1961) studied the synergism of DAB feeding and
skin painting with 3-methylcholanthrene (MC) or 4-nitroquinoline N-oxide
(NQNO), none administered in amounts individually capable of inducing
tumors. Three months of 0.5 g daily oral doses in food of DAB failed to
generate hepatic tumors, but the same treatment followed by six months
-------
on a no-DAB diet and twice-weekly painting with MC or NQNO produced one
hepatic tumor (of eight surviving rats on day 420) from MC, and, from
the NQNO, four tumors in 23 rats dying between days 160-420, and two
tumors in three rats still alive on day 420. Treatment of six months
painting by MC or NQNO followed by three months of DAB feeding produced
no tumors in the liver.
Miller and Miller (1961) tested the tumor inducing abilities
in rats, against DAB controls, of some hydroxy and methoxy substituted
4-amino-, methylamino-, and dimethylaminoazobenzenes. Dosage was
equivalent on a molar basis to 0.06% DAB. Table 107 contains the results.
TABLE 1«7.
Tor lNcim;vcis OF TUMORS IN RATSFKD THE S.UYDROXY (1IO-) mi 2- OH S-METIIOXV (M«O-)
Dr.uiv.vnvEs OF *-AMINOAXOUBN/.KNE (AH), VMONOMM JIYL/VMINOA'/OBCNZUNE (MAB),
OR 4-DlUimiYLAMINOAZOilEN7.KNK (DAB)
Kxr.
KO.
1
'
:!-Me()-AB
S-^cO-MAB
i Av.
Av. |
WT,
IM-
TIAk
WT.
COM.)
237
si;u
S-MeO-UAB 2!0
2-.V.O.AB
! -J-MeO-MAB
! iJ.McC-PAB
] a-HO-DAB
DAB
8'* ' .l-Mt-O-AB
:,
4
:! \\t-1t All
All
!Ni,hr
DAIt
•M1O-MAH
'
'2 (')
sJH
Q-US
AT
3 WK«
20
U
21
33
21
i:it ID
23.1 ! 27
233
2:15
4
25
Mill
'KH
Jv'l>"l
Vil'i
•2<)1>
TlltB
CPU.
ri.ti
8
S
8
8
8
8
8
5
H
•n H
IK
!U
77
HO
H
H
r,
»
t
i
ShX
M
M
M
M
M
A[
M
M
M
F
!•'
1-"
M
M
Sow
VIVAL
AT
4 MO.f
9/10
12/18
i
\O. lUTfc W.'TH MAUUNANT Tt ',^OH^ OF T«L:
Liver
S« Oi
pso.) i«o,)
0 1
0 0
13/in o o
6/10 ! 0 0
u/io o o
', Smiill
Ii.ttr duf~f i inl^S"*
(fl (t> (11
mo.) nm.) mo.)
107
2 0 10
057
000
000
If/16 i 0' 0 000
15/16
15/16
11/13
If/20
IS/l/5
11/15
0 0 | 0 0 0
10 13
0 0
0 Oft
0 (Iff
11 Off
i
Ml/18 0 l£tf
000
007
line
in
mo.)
:j
1
0
0
o
0
0
0
.T
i
1 7 1 Oft
0 (1
0 0
(1 0
(»tt
oft
0+t
o/io o oft o o j oft
i !
)
k'Sallil
Skill
(0 (0
mo.) mo.)
0 0
0 0
0 0 "
N,H of four, ft>\>r, ami five ruts wore foil the bnvil \|)i":iii'-i)t wits M.I n;> in ,i n-vtilltif lln- Io:v4 of fdiirnf it \(ccn n:Nfcd fl-iiM'Diovy-AII ilnrinf! I lie first :t weeks nf '"lie fif-I rxperi-
tncr.l, !'i" >iii|>i>nnit. >n,rr !!»•:(• n|>j>ei>r''ii l« be m> ilif.
f.-r.-N.d iMJm- r;i'i fcil ll.cM! Ilirce d"-l-( by .1 wi-rki, II <• i>nitn;tli fr,.-ii til! Iliref i;r'i"niii MIT<- f.-il Idu basnl did •(•• -' -
" M'-Ms'j*!'*" itir I lie ivi'mfM'Irr wcr<« HMcil.
Reprinted with permission from Cancer
Research 21:1068-72 (1961), Copyright by
Cancer Research Inc., and the American
Association for Cancer Research. 159
-------
Grice et al (1961) fed rats for 65 weeks on a diet containing
0.3, 1.0 or 3.0% of Ponceau 3R, a commercial dye consisting of a mixture
of many azo components. Hepatic tumors were present In 2/24 rats at the
1% level, and 7/23 at the 3% level.
Terraclnl and Delia Porta (1961) fed hamsters DAB, thrice-
weekly stomach tube 10 mg doses for three weeks, 5 mg i:or the next seven
weeks, and 10 mg for the last 32 weeks—survivors receiving 1.155 g.
Other animals received 3'-methyl DAB as 0.064% of their diet for 27
weeks, then 0.1% for 11 weeks—survivors receiving about 1.4 g. There
were no hepatic tumors seen by 48 weeks after cessation of treatment
with DAB. One hepatic tumor was found 23 weeks after cessation of
treatment with 3'-methyl DAB. Fifteen animals of each sex had been used
for each azo compound.
Brown et al (1961) prepared all of the 4-dimethylaminoazo-
quinolines (Q) and quinoline N-oxides (QO). Initially all of these were
fed to rats, except the 2-quinoline isomer, at 0.03% of a low-protein,
low-riboflavine diet along with no-dye, 0.03% and 0.067» DAB controls.
The more tumor-active compounds were then given at the 0.01% level.
Results are given in Tables 108 and 109. The number following Q or
QO refers to the position of the quinoline or quinoline N-oxide which
bears the azo linkage.
160
-------
Table 108
a of quinolino dyes in relation to DAB*
Incidence of li\7et tuaiorst
Code 4 Month'; G Months
0/i 0
0/10
6/10
10/11
O/'.O
l/'.O
0/10
10/10
10/10
0/10
0/10
0/10
0/10
Control (no d'~e)
DABJ (O.OtiSo)
DAB
3'-M
-------
Page deleted due to copyright clearance difficulty.
162
-------
Deleted due to copyright clearance difficulty.
Burkhard et al (1962) synthesized 2'-, 3'-, and 4'-methylthio
DAB, also 4-methylthioazobenzene. These were fed as 0.06% of the diet
(except 0.03% in the first two weeks for the relatively toxic 4'-methyl
DAB) to rats. No hepato tumors appeared after 23 and 25 weeks of feeding
the 2'-Me-S-DAB or 4-Me-S-azobenzene, respectively. After 16 weeks of
feeding 3'-Me-S-DAB 16/19 rats had these tumors, and after 20 weeks of
feeding 4'-Me-S-DAB 13/16 rats had tumors.
Weisburger and Weisburger (1963) reviewed th2 pharmacodynamics
of carcinogenic azo compounds, pointing out that metabolic "activation"
by the host animal seemed to be required, and interspecies differences
in efficiency in doing this probably accounted for part of the relatively
small number of species susceptible to azo compound carcinogenesis.
-------
Brown (1963) fed rats 0.03% of 4-dimethylaminoazoisoquinoline-4,
-5, and -7, also -isoquinoline-N-oxide-5. After four months there were:
no hepatic tumors from -4 and -7, 7/7 from -5, and 1/10 from -N-oxide-5
(all 10 died In one month). After six months there was a 100% incidence
of tumors in -4 and -7 with toxicity showing up from the latter. Retesting
of -5 and -N-oxide-5 at 0.01% level produced 0/7 and 8/8 tumors, respec-
tively, after four months, and 1/7 tumors for -5 after six months. In
Deleted due to copyright clearance difficulty.
Schmahl et al (1963) found that 233 daily doses of 33 mg/kg
of DAB, or of 3 mg/kg of diethylnitrosamine (DENA) were sufficient to
induce hepatocarcinogenesis in rats. However, on giving these compounds
together in the diet at the mentioned daily dose, the time to generate
the tumors was reduced to 153 days. Although animals fed only DENA
gained much more weight than those fed only DAB in the rtiet, those on
the combined diet had a weight gain-time curve almost superposable on that
16U
-------
of DAB alone.
Silva and Brandt (1964) demonstrated that 1,2'-azonaphthalene
was effective against transplanted Walker 256 carcinosarcoma in rats.
Both i.p. and i.v. injections worked, but best results came from using
both methods of introduction.
Manchon (1965) reviewed data relevant to food dyes.
Huggins and Pataki (1965) investigated the ability of pre-
administered azo compounds to protect, against tumor genesis by 7,12-
dimethylbenz(a)anthracene. A 20 pg dose of Sudan III was most effective.
Druckrey et al (1965) determined that weekly s.c. injections
into rats of 50 mg/kg of azoethane would produce a variety of tumors
in 37 weeks. Doubling the dose only reduced the induction time to 33
weeks.
Hampshire et al (1965) prepared some 2,4-diamino-5-arylazo-
pyrimidines and determined their toxicity to rats and mice, antitumor
activity against Murphy-Sturm lymphosarcoma in rats, and inhibition of
rat hepatic folic acid reductase. The compounds used are given in Table
112 (except for III), and the test data in Table 113. The mouse toxicity
LD-50's are for a single i.p. dose and a 21-day observation period. The
rat toxicity was determined during the anti-tumor testing which consisted
of five daily i.p. injections starting five days after tumor implantation.
There was no correlation between the enzyme inhibition and the tumor
repression.
165
-------
Reprinted with permission from J. Hed_._
Chem., 8:745-49. (1965). Copyright
by the American Chemical Society.
T• :,!•; ill.
2,!-! i', i'U'-'Q Ivb^jfvy.i! .'••> .
MIL
Hu,
[I
HI
IV
V
Y'
VII
VHI
IX
X
XI
xn
XIII
Xlf,
Cl
a
XH(
MI,
K!!,
MI,
NKti
XKU
Mis
Mi,
MI,
M
COOO;E[S
(".•x'ltcinAMi
rl
(-.(»lt';! id
tu.lIC,!^,,
COOC If,
COXHOHOTMI
NKt,
NV ifi'^IjCI),
N,C MJH.ttf).
X1S' MI] N(lit;i.'!
XV /~\,ni CH.OH (J')OKt
s /
XVI N' KfH.CH.i.l-t'n-JH.W (JOUI'Jt
TABLE 113.
AXTITl'iSOK A,\"U FdLIC Acl!> Ji£li"or.\->*: DAW
N -..
II
IV
"\"
Vf
% ; ;
vin
IX
X
XI
xn
x;:i
x :v
"X V
A -,' £
n'l"'
<;1(KK)
>!«)()
5(X)
>100l)
ii)
-!'.;<)
>10CO
240
200
170
>12U»
l.^O
Hi,( tu*k-ay,*
IBK./V.K. ri>t. a-9
SO
>4UO
> 1 !X)
220
>2l)i)
>2U«>
>•!(", 3
140
Dose, » ;.
W4-. ''K- >!• COO
166
-------
Fare (1966) painted the dorsal skin of rats (1 ml) and nice
(0.2 ml) with a 0.2% acetone solution of some 4-amino azobenzenes twice
a week. Results are in Table 114. The compounds were chronatographieally
pure.
TABLi'I IIH-
UKSUI/W cit- l*,\iN'its«; SKIS «»v lt.vi> \N» Mu'B \\ITU A?,<> DVKS
SrivtES
Hat
»nt
Rat
lint
Stx
M
M
M
M
Kat M
Hut
Hiit-
Mouse
Mouse
M
M
F
M
Dve
None (Control)
AAB*
MAB
DAB
3-MeO-AAB
3-McO-MAB
3-MeO-DAB'
3-Mt>0-!>AB
3-MeO-DAB
MFAM TIMK
tM LWUIN
tant.u («k.)
i!7
44
73
7(i
28
47
MKAN UW.TM
131
123
58
TO
03
41
(12
30
G2
I
Tl MO* IM llll.Si'K
Kur iluct
«/(»
0/0
o/<;
o/c>
J/H
3/0
:i/lo
0/1 -ID
0/1 10
.Skin
0/0
tt/fi
('»/*>
(i/ti
li/W
c./o
K//10
0/140
0/140
TOTAL YUtn ti^ SKIN T^'HOHS i.s r.te'»rt», INISTUOMTI u'
Kpider-
moi'l
cyst
5
ill
2
3
27
2
Krntlo- I •
ihuma
s
1
a
a
4
7
3
3
12
10
1
.,„„.
&
18
11
5
28
10
AnapLt^lif
2
9
4
5
13
1
Si! i,\
|.,i|.ll
U i,j
1
a
2
2
1
MlM,
3
7
3
5
u
4
0 Tumor types are described more fully in tlw
'Abbreviations: AAB, aminoazobcnxene; MAB, monomethyltunimw.obcnzeiie; UAB, dimcthylaminoMobcnzenc; MeO-, mc-liioxy
group.
* Data from Fare and Orr (4). The uumbcre of tiiiiuim prudiicod arc not oompiiniWe with those in the other treatment, groups
.•Ninrc in this particular case nut all tumors wore examined histologtrally.
Reprinted with permission from Cancer^
Research 26:2406-8 (1966). CopyFTgFT by
Cancer Research Inc., and the American
Association for Cancer Research.
Kanekar and Pause (1966) force fed rats five times a week
with 0.4 ml of a 1% peanut oil solution of technical grade, or 0.4 ml
of a. 1% suspension in normal saline of purified 2*, 3-dimethoxy-4-and.no-
azobenzene. A tumor appeared in the skin at the external auditory canal
opening after 156 days of dosing with the technical grade, and 174 days
of the purified compound. Other rats survived 242 and 280 days, respec-
tively, before developing this (and no other) type of carcinoma. These
carcinomas were not transplantable.
Brown and Hamdan (1966) prepared and tested in rats some more
nitrogen-heterocycle/azo/dialkylaminobenzene compounds. The results are
16?
-------
in Table 115. Except for the last three entries in the cable, P04 is
an abbreviation for 4-[[p-(dimethylamino)phenyl]azo]-pyridine,l-oxide;
using this nomenclature the entry above P04 itself, e.g., should be
written 2,3'DiMeP04. In the last three entries the dimethyl of. (dimethyl-
amino) has been replaced by the indicated alkyls.
Table 115. Rat Hepatocarcinogenesis from
Alkylaminophenylazopyridine-N-oxides
Compound code
Control
DAB
DAB
2'McPOl ,
2',(>'DiMeP04
2'Mol'CM ...
2' G'DiMcPCH
2'MoPOl
2' G'OiMcP04
22'I)iMeP04
2' 3DiMcP04
P04
McKtPO4
DiJOtPO-1
DiPrPCM
Percent
in diet
0.06
03
03
03
02
02
01
01
1 03
03
03
03
03
03
Tumor incidence (months)
2
0/10
8/10
3/10
4
0/10
7/10
0/10
7/7
4/4
10/10
10/10
10/10
9/10
7/10
8/8
10/10
8/8
0/10
6
0/10
6
0/10
9/10
5/10
10/10
0/10
12
0/iO
10/10
0/10
Polrier et al (1967) prepared N-benzoyloxy-N-methyl-4-amino-
azobenzene and tested its reactivity with various biochemicals, also
its ability to generate carcinomas in rats after s.c. injection. Their
results are in Tables 116, 117, 118, 119, and 120. Some closely related
azobenzenes were tested'for comparison.
168
-------
TAW.K tlfc.
of X-licnz<»/l<>jit-X-iiirlliiil-4-tit»iri<><'n2r.nc itml Itrlalfil ('i»iifnniml*
AtliinnixIcFc'l /;// AV^fu/f:*/ $.c. ftijccttunx r/i I'nlx
Kuril nil was injected s,c, in the right hind leu twice weekly willi 0.2 ml of triorltmoin in which the
lost compound had been dissolved or mmpr»de,d \\ tlhmil heal immedmU ly prior to injection.
Compound
Kxpprimoni 1
A'-Beiizoylnxy-
MAB»
MAB
N'one (vehicle only)
v
Experiment 2
A'-Bciixoyloxy-
MAB
MAB
\
.V-Beiwoyl -MAB
A'-Hydroxy-AB
AB " \
DAB-.V-oxide
DAB
None (vehicle only);
j
nose
24 X .3.9 mg
24 X 2.5 mg
24 X 0.2 ml
24 X 3.9 mg
24 X 2 5 mg
24 X 3.7mg
~24 X 2.5 mg
24 X^2'.3 mg
24 xV0 mg
24 X 2,7 mg
21 X 0.2 ml
No. of
faK anil
10 M
10 F
10 M
10 F
10 M
10 F
20 M
20 M
20 M
20 M
20 M
20 M
20 M
20 M
AvcrnKe
Ht It >-. ccks
-------
TABLE 117
of Huh ctnti On'an cute of Tiiiinirx after i.p. Injcclitmi of X-AIr,lli!/l-4-ii>itifi»iiiiilM'ii*fnr
(MAIi) at (U 'X-Hcnzuifloty l>erivulive into Nftmnlul Hals
Ompoumt
Kxperimont I'1
A'-Bi-n/.m]-
oxv-AIAll
MAB
Corn oil only
Experiment 2C
A'-Beiiinyl-
oxv-MAH
MAB
Trioctanoin
only
Tol.il
1.00
0.65
0.48
».30
No,
46
47
33
162
101
\
*
110
No. alive
at U (lays
11
30
25
98 '
73
\
X '
71
No
and M.-X
7 M
4 F
8 M*
8 F*
8 M»
8 F*
SO M
42 F
40 M
33 F
X "*•
32 M
39 F
7 mo.
G
4
8
8
8
8
55
38
40
33
32
39
So. of survivors at
10 mo.
4
4
8
8
8
8
48
32
39
33
32
38
13 mo.
4
3
8
8
8
8
28
25
34
-
32
32
38
19 mo.
4
1
7
7
5
4
8
9
24
23
21
35
Nf*. f)t r.itv vvilit £rQ£S
lumors by 1'J mo.
1, bilutcial renal rurci-
noina.H (7 mo.)
1, ni'illiple pupillomits
(urinary blndiler) (I!)
1, bilateral renal carci-
noma* (12 mo.)
1, mammary gland car-
cinoma (19 mo.)
0
2, mammary gland ade-
nomas (1'J mo.)
1, carcinoma in silu
(skin) (19 mo.)
0
1, pulmonary adenoma
(17 mo.)
3, pancreatic adenomas
(l.'J-lo mo.)
2, sarconuts (injection
site) (13 and 14 mo.)
1, renal carcinoma (7
mo,)
1, biu^al cell carcinoma
of lip (l!l mo.)
1, ehnlaiiKioma (1!) mo.)
2, mammary gland adc-
nonia.s (19 Inn.)
1, ean"ii!«!mn of ear-duct
gland (13 mo.)
1, clmlangionia (1!) mo.)
1, Iciomyoma (small in-
testine) (19 mo.)
2, cutaneous papilloinaa
(lil mo.)
1, malignant lymphoma
(14 mo.)
6, mammary gland ade-
nomus (17-19 mo.)
1, mammary gland car-
cinoma (15 mo.)
1, cholangioma (17 mo.)
1, sarcoma (foot) (17
mo.)
1, culaneons papilloma
(19 mo.)
1, mammary gland car-
cinoma (8 mo.)
7, mammary gliind filmi-
nilriH.in:iM (15 1!) mo.)
" I'liwli nil WHM injected i.p. with 0.05 nil of MleriU1 corn oil idono or containing 0.2 nij.; of .V-henxoyloxy-
MAH or 0.13 lug of MAB within 24 hr aflnr birth and on each of the sncrcediiiK 2 days; on the 4th day
the rain wore injected with 0.1 ml of ihc .same solutions.
* Because of I he poor survival of the1 ral» which received injeclioim of A'-lviMoyloxy-MAH in this
preliminary cxiwrinicnt, only 8 nuilc and 8 fcinulc rul.s of the 30 und 25 ralK injected with MAIi or corn
oil were kepi ut weaning,
* Kach rat wan injected i.p. with O.Oo ml of sterile trioetanoin alone or containing O.Jfi mg of A"-ben-
zoyloxy-MAB or 0.10 mg of MAB within 24 hr after birih and on each of the succeeding 2 days.
170
-------
TABLE liy.
The Reaction of X-Uenzoytory-'S-niftli!il-4-I>rmrne and
Other Aminoazo Dyes irtth liorinf Scrum Allmnun*
TABLIC
jV-Ueiusoyloxy-iV-meihyl-
4 -uininoazobcnzciie
jV-Mclliyl-4-amiiwazo-
benzcne
.V , N -Dime t hyl -4 -am i no -
benzene •• ,
4-Amim>iW.obenzeiie
A', A' -Dimethyl -4-amino-
ti r,obonzcnc-JV -oxide
A' Slyilroxy-4-aminoa7.o-
Il("II?,l'll(!
A'-n.vdroxy-,V-a<:cly]~t-
amiiio;w»benzcnc
(.ibsorbance/SO ms protein)
Polar fraclioo
1.11 (520 HIM)
<0.01
<0.01
<0.01
0.01
<0.01
\
<0.01
Nonjvolar fraction
0.-10 (507 n>M>
0.01
0.01
0.02
0.02
<0.01
0.01
« ]kivine scrum albumin ,(125 m«) was, incubnred at pH 7 with
1.0 nig of A'-benzoyloxy-2V-metbyl-Kiimino&«>l>en/.cne or an
equivalent amount of .another dye for 4 hours at 37*0 in a nit rogon
atmosphere. The analytic procedure is described in the Materials
ami Methods section. The analyses for each of the polar and non-
polar fractions were corrected lor Wank values of 0.02 which were
obtained when serum albumin was curried through the .sumo pro-
ri'diiri' in dm nlwfiiiue, of liny dye.
'The (inures in piironlhosfis sire flu- wave lengths of maximum
absorption for the dye fruetioim derived from AM>ciizoyIoxy-JV-
methyM-iuiiinoazobenzcne; the corresponding fractions derived
from tlt«" other dyi'H showed only low nbHorbiMiccH nnd for wm-
v«*i» ScivglliH Hhowii,
TABLE 1ZO,
The Ittartioii of X-Iicnz0iilox]i-X-mclktjl-4-aminoazobenzcne
(S-iicn:oylory-M,\O) und Other Dfu wilk Variant Nu-
chosides*
lir*n-
incat
.\a.
1
2
3
4
0je
Ar-Be»7,oylosy-MAB
MAU
A'-l Iydroxy-4 -ami no-
axobcnzeno
.V-Br nzoyloxy -M A B
A'-Uenzoyloxy-MAB
.V-BenzoySoxy -> IAB
A'-Bonzayloxy-XIAB
Ar-Bcnzoyloxy-MAB
A'-Bcnzoyloxy-MAB
.Y-Bcnzoylosy-MAB
A'-Bcnzoyloxy-MAB
A"-lionzoyloxy-MAB
Kvcicoside
Gt>a«asinc-8-I4C
Guatws"me-S-1'C
Guaoosine-8-"C
Guanosinc-8-"C
A«irinn»
% reaction j m,ii;m*Mi .'i
at KB nun6 ; »jlrr -nlJ..f
•"
ilieubiiteil in a nitrogen almnspliorc at 37"C for 00 mill. Kxccjil I"!
the product forr.ied from mcthioninc, the extent of reaclimi »;i-
dctorntincd from the amount of water-solulile dye whirh rctii'ii'"'!
after extraction with beozene-hnxnne.
'The % rcnilioii was oilt'iilaied on the assiiiii|)tion thiit ill*
produets hud the sume molnr absorpmin roelliciPittM «s MAll \
hlatik eqnivftlcnt to a reurtion i>f I 5*V wan siil>triieli*d from '•>'•'
Viiluos bawd on wstter-Milublo dye whoit tin' muiim iicids V.ITI- u
cilbated willi A'-ben/.oyJnxy-MAli. A lilmik i>f 0.0' , \va,-i tiliUm- '
when MAB was incvibulod anil cxlrncu'd «?> deHrnbfil.
' The Milfimiiim dcriviilive formed from melhiouim' ui,>dn,'ili'
'docomposes even jit neiitnilily (see Oharl 1). Tlierelore, 'I'1
amount, of 3-nie()i''lnieix>aplo-MAU formed >.puiiliine(iu.--!y jili.
tluil fonitiMl after (lit* mliSUton uf nlUiili w it ln%uer estiiiMlc of ''"
exlenl of reuelion lliull (lie inuouiil of wider milul'lo the.
Tables 116-120 reprinted with permission
from Cancer Research 27(9):1600-13, 1967
Copyright by Cancer Research Inc., and
American Association for Cancer Research.
n
-------
Odashima and Hashimoto (1968) reported the following results
of a 60-week feeding study on male rats. At a level cf 0.08% 4-amino-
azobenzene (AB) caused one peritoneal cavity tumor in 1/32 animals.
At the 0.09% level 3-methoxy AB caused hepatocarcinoma in 21/23,
malignant splenic hemangiopericytoma in 6/23, and squamous cell carcino-
ma of the ear duct in 2/23 animals. At the 0.1% level 3,4'-dimethoxy AB
caused hepatocarcinoma in 24/24, squamous cell carcinoma of the ear
ducts in 1/24, and tubulary adenocarcinoma of the small intestine in
2/24 animals. At the 0.09% level N-methyl AB and N,N-dimethyl AB
caused only hepatic tumors, but did so more quickly than the other
compounds.
Brown and Sanchorawala (1968) found the following incidences
for hepatocarcinogenesis in rats at the indicated dietary levels:
0.06% DAB-70% at 4 and 90% at 6 months, 0.03% DAB-50% at 6 months,
0.03% 3'-methyl DAB-50% at 4 and 90% at 6 months, 0.03% N,N-dimethyl-
p-(6-benzothiazolylazo)aniline-50% at 1 and 100% at 2 months, 0.03%
N,N-dimethyl-p-(4-benzimidazolylazo)aniline-100% at 2 months, 0.03%
N,N-dimethyl-p-(7-benzimidazolylazo)-aniline-100% at 3 months, 0.06%
N,N-dimethyl-p-(4-benzothiazolylazo)aniline-0% at 6 months, and 0.03%
N,N-dimethyl-p-(5-benzothiazolylazo)aniline-0% at 6 months.
Druckrey et al (1968) exposed 15-day pregnant rats for one
hour to 4800 or 9600 ppm of azoethane, equivalent to 300 or 600 mg/kg
(14 or 28% of the LD-50). At the lower dose 41/42 animals developed
multiple neurogenic malignomas including 25 in the brain, 20 in the
spinal cord, and 29 in the peripheral nerves. The corresponding
numbers for the higher dosage group were 28/30, 16, 6, and 24,, The
172
-------
latter group also showed malformations of the paws. Tnere may have
been tumors in the dams, but the authors did not break out those tumors
from azoethane (if any) and those from some related non-azo compounds
also tested; in 32 dams there was a total of seven tumors.
Clayton et al (1968) fed rats a diet containing DAB deriva-
tives at the molar equivalent of 0.06% DAB. All animals had hepato
tumors at 9, 22, and 22 weeks in groups fed the 3'-methoxy, 3'-cyano,
and S'-acetylamino derivatives, respectively. All three caused
severe cirrhosis in addition. No tumors resulted from 26-week feeding
of the 2'-, 3'-, or 4'-carboxy, 2'- or 4'-methoxy, and 3',4'-dichloro;
the 3',5'-dichloro produced one tumor in 12 rats at 26 weeks. In a
follow up study for comparison with 3'-methyl DAB the uhree active
compounds were fed for 51 days, then removed from the diet for eight
weeks. At this time the percentage of survivors having tumors was:
3'-methyl 70, 3'-cyano 77, 3'-methoxy 85, and 3'-acetylamino 90.
Brown and Snider (1968) prepared an additional seven
dimethyl-substituted derivatives of N,N,dimethyl-4-(4'-lpyridine-l-
oxide]azo)aniline, designated P04', in addition to the four reported
on before. These were fed to rats at 0.03 and 0.06% of their low-
protein, low-riboflavine diet along with 2'-methyl P04', 3'-methyl P04',
and DAB, all for comparison. The results in generation of hepato
tumors are given in Tables 121 and 122. The relative activities of
the various compounds changed with the dietary level given.
173
-------
Table 121
•Tumor incidences (0.03% level)
Tumor Incidence (months)
Compound code
DAB
2'-MeP04'
2,3-l>iMcP04'
2.3'-l)iMcP04'
•J',.ri'-l)iMc-P04'
'1 .VDi.McPOl'
2>])iMcP04'
S'-McPOl'
3,3'-UiMcP04'
3!,5'-DiMeP04'
4
0/10
10/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
5
0/10
0/10
6/10
0/10
0/10
0/10
6 8
6/10
0/10
0/10
2/10
0/10
0/10 10/10">
0/10
0/10
Table 122
-Tumor Incidences (0.06% level)
Compound code
Tumor Incidence (months)
3466
DAB
2,3-DiMcPO4'
2,3'-DiMeP04'
2',5'-DiMeP04'
2,5-DiMeP04'
2,6-DiMeP04'
3'-MeP04'
3,3'-DiMeP04'
3',5'-DiMcP04'
7/10 0/10
0/10 0/10 0/10 10/10
0/10 10/10
0/10 10/10
0/10 0/10 10/10
0/10 0/10 0/10 10/10
0/10 10/10
0/10 10/10
0/10 0/10 10/10
Brown and Fisher (1969) prepared and tested for hepatocareino-
genicity in rats the following compounds at the 0.03% level: N,N-dimethyl-
p-(3-,4-»5, or 7-indazolylazo)anilines—no tumors after 8 months; N,N-
dimethyl-p-(6-indazolylazo)aniline—100% effective after 5 months;
17U
-------
Deleted due to copyright clearance difficulty.
17';
-------
N,N-dimethyl-p-(2-, 5-, or 6-quinoxalinylazo)anilines showed no tumors
after 8 months, 100% tumors after 4 months, or 100% tumors after 2 months,
lespectively.
Bebavi et al (1970) prepared some new DAB derivatives,
substituted in the 3',4' positions and compared them with DAB and 3'-
r.ethyl DAB for rat hepatocarcinogenicity after 50- or 365-day force
feeding. Their results are in Table 124.
Table 12.4.
Carcinogenicitiet of the DAB's
A. SO days of dye administration
DAB
Percentage of rats with hcpatomas
after start of dye administration'
R-3'
H,-
Cl-
U -
. '!,-
( ..Us—
C.H,-
L'.'Ha—
H
CH, -
CH,-
R-4'
Cl-
CH —
Cl—
CzHs—
CH,—
C;IU—
H -
H-
CH.t—
H—
Dose*
0.50
1.00
1.00
0.25
1.00
0.50
0.50
1.00
1.00
0.50
B.
4 mo.
50 (50)'
0(10)
0
0(15)
0(60)
0
0 (10)'
0
0
10 (50)
Continuous dye
8 mo.
75
. 0 (10)'
0
15(15)
0(10)
0
0
0
0
50 (30)
administration
12 mo.
too
0
0
40(10)
20(10)
0
0
20
0
60 (20)
• Activity
rank'
4
0
0
3
1
0
0
1
0
?
DAB
Percentage of rats with hcpatomas
after start of dye administration'
R-3'
CH,—
Cl-
CI--
at!-
OH,-
C,H5-
CHU-
H-
CH,—
Clli-
.R-4'
Ct-
CH,—
a—
C,H5—
CHv-
C,H»—
H —
H—
CH,—
H-
Dosc"
0.50
1.00
1.00
0.25
1.00
0.50
0.50
1.00
1.00
0.50
4 mo.
40(60)
0
0
40 (25)
30 (30)
17
10
25 (30)
10 (40)
90
6 mo. 7 mo.
100
75 100
20
50 100
100
8 mo.
80 (20)
40 (30)
90
33
60 (30)
75(15)
12 mo.
100
100
100
67
100
100
Activity
rank'
8-
2
1
10
4
5
6
3
7
9
Reprinted with per-
mission from Cancer
Research 30:1520-24
(1970). Copyright by
Cancer Research Inc. ,
and the American Asso-
ciation for Cancer
Research.
" Doses are expressed as fractions of "1 dose-equivalent," whicli was equal to 0.0375 ninioles/
day. The daily dose was administered in 0.5 nil corn oil except for 3'-CHi -, 4'-CI—, 3'-C.l--, 4'-
CHi--, _V,4'-DiCI—, and 3',4'-DiCH,-DAB's which, because of lower .solubility, were ;irlmmis-
tered in I ml.
' Percentages are used because groups varied from 10 to 20 animals. Occasionally, u rut died
following a laparotomy; it was then not included in the percentages after that time.
'Activity or carcinogenic rank was based on the incidence of hepalomas and consideration of
the quanlil) administered (dose equivalent). The larger the rank number, the greater the car-
cinogenic potency
' Percentages of animals with livers that were not normal in appearance at the time of regular
laparotomy but which were not scored as carcinogenic.
'This animal died 2 days after the 2nd laparotomy with a cirrhotic. nodulated liver, but it was
scored negative.
'This animal died before 2nd laparotomy; a cyst was found in liver, it was scored ncgulivc.
176
-------
Child et al (1972) reported on the anti-cancer activity of
some polyhaloazobenzenes in mice. Mammary adenocarcinomas were
transplanted into mice and allowed to grow for 17 days. Then six daily
i,p, injections of the test compound were given, followed by excision
and weighing of the tumor. Table 125 gives the results, C/T ratio
being that of the tumor weight of controls to test mice. Compound
(d) was considered marginally active, the others fully active,
TABLE 125-
Pooled result* obtained with dosc-rc>sponse tests compounds
administered Inlrapcrltontulty dully
Compound
(c) 3,3',1,4'4rtriil
8F0tH'l**t'W
(d) frliyilinxy 3.1
trUiu'Iilf'Mf t/ol
(o) 2,2',^,3',4,'l'-ti
chloiu imU-ft'tt'
Doso
tit
mg./l'K.
260
nn 250
MO
I',4,4'-
ii'itn'iie-. ?50
ota-
HO 250
Number
«(
poups
tostud
3
2
3
4
2
Number
of
Bnlmals
27
18
27
at
IS
Averoce
C/T
activity
ratios
4.63
S. 92
4.74
3.40
10.10
In Table 126 are the results of the same type of experiment, except that
an optimal dose and survival time were determined; the figure in the
last column includes the 17 day pretreatment, tumor-growth period.
TABLE izfc-
Results ot survival studies In tnico twatUig transplanted mammary &de-
nocjirclnonia—Administration lntr.iperitone.iiiy onco djily
Median
»urvlr»l
Op!!- 'limes in
Jnally Number Num- days at
«ffecUvo of bero( opiiuulty
dose in groups anl- eHcciivn
Compound mg./kg. tested mals dose
S.a'.-iJ'-lotreehloroazobwizcne-. 8i 3 W 86
3,3'*dichioro-l,4'-ilHiuoro32o- 9 2 60 38
bonzene.
2,2',3,3',4,4'-hetiu:hloroazabea' 3 1 10 34
ft-lie.
Brown and Kruegel (1972) prepared the six trimethyl DAB deriva-
tives (all three methyls in the "prime" ring) and tested them against DAB for
17?
-------
rat hepatocarcinogenicity. The 3' ,4' ,5 '-derivative was equivalent to DAB,
but the others showed no activity in nine months of testing.
The International Agency for Cancer Research Working Group on the
Evaluation of Carcinogenic Risk of Chemicals to Man has been preparing mono-
graphs on the following list (finalized in June 1974 — private communication)
of azo compounds.
Table 127. Azo Compounds Undergoing Evaluation by IARC
DYES
C.I. Acid Orange 10; 1936158 ', 1-Naphthalene azo-2',41-
diaminobenzene; C.I. Food Orange A; Orange G
C.I, Acid Orange 20; 523444 ; p-/t4-hydroxy-l-naphthyl)azo/benzene
sul phonic acid, sodium salt; Naphthol Orange; Orange I
s
C.T. Acid Red 2; 493527 5 o-/"/p-(dimethylamino)pheiiyl7azo7benzoic
acid; Methyl red
C.I. Acid Red 14, disodium salt; 3567699 ; C.I. Food Red 3;
2- (4-Sulpho-l-naphthy3 azo) -l-naphthol-4-sulphonic acid ,
dj sodium salt; Carmoisine
C.T, Acid Red 26, disodium salt; 3761533"; 3~Hydro\y~4~(2,4-
xylyazo) -^//-naphthalene disulphonic acid, disodium salt;
Ponceau MX; Ponceau 2R
t;.I. Acid Red 27; "915673*; 3-Hydroxy-4-/(4-sulpho-l-naphthyl)azo7-
2 ,7-napbtba)one disn] phonic acid, tri sodium salt; Amaranth
(', f. Basic Oiar.p.e 2; 552821 ; 2,4-DJaminmzobenzenc-4-
hyih-ochlorick*; C.I. Solvent Orange 3'r ChrysoidJne . -
:,.':." jiiieri ]5Juc-14-;-- 72571- ;:-_75,3--/(3;3l
!>i;iher.y]yloui''jbi5; - (rr. oi7bis/5-ai'UriO-4-li
.;, <\ >>it] ia it iiciuisxt] phonic acid, tetrasodiuni salt; Trypan Blue -
C.i, Direct lUne 53; 314] 36 j 6,61-/(3,3'-Dimethyl-454 ' -
li .;.f)--"r,'l y i - ;v)i)if;(;r'c;)7t< ' f./4-aniino--.r.-hydro>:y-l ,'''•-
(•III )i.') h »' ') i .snl phnnj' ;K, id ietja sodium s;i)l/; Kvans JUuc
|)I'.|MI i V.llt.u* '•; /,H,',/ i()i! ; 4» /ft)-!lyi!i('.,y m Idyl Jn.-u/
1 78
-------
C.I. Pood Red 1, disodium salt; 4548R32 ; 4-IIydroxy-3/(5-sulpho-
2,4-xylyl)azg/-l-naphthalcnc sulphonic acid, disodium salt;
Ponceau SX; "~1T> + C Red No 4
C.I. Food Red 6; 3564098 ; 3-]Iydroxy-4-/(2,4',5-trime1.hy]pheny] )
azo7-2,7-naplvthalenedisu]phonic acid, disodium salt; Ponceai 3R
*
C.I. Food Yellow 3j disodium salt; 2783940 ; 6-Hydroxy-5-/(p~
sulphophcnyl)n?.o/-2-naphthalcnesulphonic acid, disodium salt;
Sunset Yellow F(iF; FD + C Yellow No 6
- *
C.I. Food Yellow 6, monosodiuni salt; 2491761 ; 4'-Chloro-4-
diinethylamnoazobenzene
C.I. Pigment Red 53, barium salt; 516002] ; 5-Chloro-2-/t2-
hychx>xy-l-naphthyl)azo7~p-toluenesu]phonic acid, barium salt;
D & C Red No 9
C.I. Solvent Orange 2; 2646175 ; l-(o-Tolylazo)-2-naphthol;
Oil Orange SS
C.I. Solvent Oranpc 7; 3118976 ; I- (2 .4-xvivlazo1-2-naphtho] ;
Sudan 11; Oil Orange XO; ;md its 2,S-j.somcr 8582.riA
C.I. Solvent Red 19; 636872S ; N-Erliy]-l/7p-(phenyla7.o)pheny]7
rizo7--2-naphtliyJaiiiine; 5iiuiiu Red 7B
C.l. Son/oil Red ?3; 85869*: 3 -/7p- f ]'];e]iylazo)pljrnyl7azo7-2-
najjr-xiiol; Su'i.i'j JJ1
CJ. S.xlveul. Rc-1 >!; 85-S?''' ; 1-//4 (o~Tolylnzo)o -tr.ly] 7azo7-?.-
- 71:in!;l};oJ ; - S.;.rii:J ilod . - -"
CJ. r^Jvrm R.-fV f'0; 63s8!,:;8' ; l-/(2,5-Din.ethoxyp!icny])azo/-2'
CJ. :• iV\.';"iT Yc"'"'.': 1; "61"'^:'') , p-Ainiii'^i/obc-nzene
CJ. Silent YeiJvjr 2; 6CJJ7 ;
Ik: I l.c-j- Yellov;
C.I. Solvent Ye'llov 3; 97563 ; o-AmJnoazbtoluene
C.J. 5 -jl vr-n t Yd !..•>.• I.; ;;!,;: IV ; ]-(r!r_'nyla7oJ-?.-iiaplilhylajiiine;
Yc ! )(>,; Ali
C J . ,(.-,lvc'i)1 YeJK',/6; ]317'i?; ; l-(o TolyJ azo)-2-jmj)]»lliyl ajui
Yd I:.-. OH
179
-------
*
C.I. Solvent Yellow 7; 1689823 ; 4-Hydroxyazobenzcne
*
C.I.'Solvent Yellow 14; 842079 ; l-(Phenylazo)-2-naphthol;
Sudan I
Other uses such as pesticides, drugs, etc.
*
Azobenzene; 103333 ; Diphenyldimide
*
Diacetylamtnoazotoluene; 83636 ; 4-o-Tolylazo-o-diacetotoluide;
Diniazon; Pellidole
*
p-Dimethylaminoben?.enediazo sodium sulphonate; 140567
*
Elaiomycin; 499489 ; D-threo-methoxy-3-(l-octemyl-ONN-azox>0-2-
butanol
*
Phenazopyridine; 94780 ; 2,6~Diamino-3-phenylazopyridine
Chem. Abstract No.
1BO
-------
C. Lower Animals
Allen et al (1957) reported that azobenzene was toxic to the cyclamen
mite Steneotarsonemus pallidus.
Hayashi et al (1960) in a study of anthelmintics found that Ascaris
lumbricoides was susceptible to 1 part in 40,000 of 4-(4-chlorophenyl-
azo)phenol(A), 4-(4-nitrophenylazo)phenol, and 4-(3-nitrophenylazo)phenol(B)
Rhabdias bufonis was susceptible, in decreasing order, to A, 4-
(4-bromophenylazo)phenol, and B. In vivo in toads a 300-400 mg/kg
dose of A was equivalent to an 800 mg/kg dose of 4-iodothymol or of
l-bromo-2-naphthol.
Iwashina (1960) published the following list of a-zo compounds and
their toxicities to earth- and intestinal worms.
Table 120, The Relative Efficacy of Various Organic Cotnpomnds
againrt Earthworm and Animal Parasites in u'.'ro.
No.
247
255
*»
265
/•
N'C
<
~\-N7 = N
»-N==N
Chemical)
(Formula)
OH
s'=N-<^OH
s-=N-'/~^-OH
O°H
t
- Minimum
concentration
to kill earth-
worms in 24
hours
1;: 40,000
1 : 80,000
I : 160,000
1 : 80,000
Minimum:
concent-
ration ten
kilt pie;
ascaris in
24 hours
1 : 20,00(0
I : 40,00(0
1 : 40,00
-------
fable T28, Continued
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
/~"CN-N -/~VOH • '•••"•
NO,
NO,<>N=N--OH-
NO,
NO,
NO,
\
NO, (f)
N°i~m Mf
Br
/
I (t)
X~™X y— »v,^
NO -^ ^-N=»N" -OH
Cl (T)
NO,-/ \-N = N-/~~\ OH
,1 80,000 » U: -fiO.Oa)
: 1 160,000 . E : M.OOO
1 160,000 C> : 5,000: > : 2,580)
1 160,000 « Es. ,'20,000
1 320,000 * C : 160,000
1 160,000 [1; 2.500J I: 2,500
1 160000 . C: 10'000
1 MJU.WU « —20,000
1 160,000 Cl : 2.500J ^ r 5,000)
1 80,000 • 11:-. 80,000"
1 160,000 Cl = 2.500J ~- = 2.500J
Cl 10.000J . JL 2,5003
1 160,000 Cl = 2,500] :: 160,000
1 160,000 Cl = 2,JOOj ; : it>0,000
1 s 320,000 1 ; 2,500 "iiJi'ooo
1; 320,000 1: 2.500 : : J20,000
(1 : 5,000]
Cl: 5,0003
Cl : 5,0003
Cl: S.OOT)
Cl : 5,000)
Cl ; 5,0003
|I : 5,000)
-------
Table 128. Continued
316
317
318 KC
319 NC
N=N-/ Von
(t)
(t)
Br
Br
Cl
-N=N- >-OH
a
/ (t)
320 NO,-/~~VN=N~<^~~\-OH
321 NO,~
>-N=N~<
Br
322 NO,< >-N = N< >OH
Br
Cl
323 NO,-< >-N=r
X
-OH
324
325
326 NO,-<
327 N'O,-
Cl
Cl Cl (t) '
Cl Cl (t)
Cl
/
Cl
/ (t)
Ct
a
ci
• \
=~"
1 : 80,000 •
1 : 320,000 »
( : 5,000 « t : 160,000 *
1 : 6-W.uuu (.r : Z.aoO) (1 : 2,500]
1 : 2,560,000 [1 : 2,500} I : 40,000 (I ; 5,000^
1 : 640,000 (I : 2,5003 1 : 80.000 H : 2,500]
I . fin «YI .
I. 80,000 •
1 : 40,000 ,. . -of.
-flO.OOO (I ' 5>OOOJ
I : 320,000 Cl : 2,500) 1 : 40,000 C1 : 2.500J
1 : 80,000 •
1: 320,000 (1 : 2,5003 •
1 : 320,000 •
1 : 80,000 r, , 5 port'
—160,000 l '
1 : 1,280,000 »
1 : 160,000 C^ : 5'00lV
183
-------
Table 128. Continued
Br Br
328
\
NO, <
i»
331 N01-
NO,
Br
NO,
NO, CI
NO,
\
Br
Cl
(f)
Br
NO,
Cl
133
"Br
(t)
>~OM
NO, (f)
\
Cl
NO,
Br
/
US NQj~
-------
Table 128. Continued
J55|
J56
557
OH-HN< >NH-HO-
-
s
0"C-CH = CH-CH = CH-CH = i
V
AH,
C.H,
29
183
231
OH
I
H
C.H.,
236 Cl-
: 2.500J
: 5,0003
1 • 4fi nnn I • w rrr ' '•
1 . 40,000 1 . 20/JJ. _i
1 : W.OOO
-80,000
1 : 20,000
1 : 5,000 .
1: 320,000 [1: S.xC 1: 10,000 [1: 5,000]
1 : 40,000 1 : 5,CO'j 1 : 20,000 1 : 2,500
1 : 80,000 1 : 40,OX 1 : 160,000 1 : 5,000
: 80,000
—160,000
: 160,000 ' 1 : 20,000
1 : 80,000 1 : 40,OX I : 320,000 1 : 80,000
[ 1 : 5,000] =not effective in the dilution of 1 : 5,000.
• =not tested.
Jeney and Zsolnai (1964) reported that Ascaris suum, in vitro, was
susceptible to these dilutions of various 4-amino-azobenzene (AB) deri-
vatives: 1/10000 for AB, 1/5000 for 2',3-dimethylAB'HCl,1/5000 for 2,3'-
dichloroAB'HCl, 1/5000 for 2-aminoAB-HCl(Chrysoidine), 1/10000 for 4'-
chloroAB, and 1/10000 for 4,4'-di-(p-aminophenylazo)biphenyl-2HCl.
Zsolnai (1964) reported that Ascaris suum, in vitro, was susceptible
to the dilutions of the various phenylazomalonitrile derivatives in
Table 129.
-------
Table 129. Ascaricidal Limiting-Concentrations of Phenyl-
azomalonitriles
I'licnvUii/n'-nvi limit ril
2- 1 olyl-.i/o-iii.iluiiiliil
VI nIyl-a/o-iliaU>l>illil
•1- 1 olyl-:i/o-malomliil
2-( hloi-phaiyl-a/o-malimiliil
3-('hlor-plu:nyl-a/o-m.iloniiril
4-( 'hk u -plu.-nyl-a/i>-i n.i li mil nl
4-l)Hiiii-plteiiyl-u/i>-iiuli>inliil
4-JiKl-plk'iivl-a/o-inaloiiilril
• l-AcllKt\v-plu'iiYl-a/o-m.iU>iu(iil
2-Mclh) I- 1-bmiii-plii n> l-.i/o-iu.ilonilnl
?.-Mi:lli>l-4-iinl-pliiinyl-.i/ivnulin\ilril
1- Mi 1 1 1> l-l-bioMi-plii'iiyl-.i/n-iii.iliMiilril
2-liiiim-4-aellH>xy-pliaiyl-j/o-nialoniiril
2,5-Oidiloi-plienyl-a/e-malonilril
3,5-Dihrom-pliLnvi-a/o-maloiiilril
2-C'lH>r-4-hrom-plicnyl-:iA)-iiuli)iiiiril
3-Chor-4-brom-plii:nyl-a/(>-ina limit ril
2-Mc;liyl-4.f)-ilibri)ni-phcnyl-a/o-m:ilinii(ril
"
3-C'liloi'-4,fi-ilibroni-plicnyl-.i/u-iiialoiiitril
4-n)liir-2.(i-ilibrom-pln.'nyl-.i/o-m,iloiutril
2,4,()-Tiibn>iii-phcn> l-.i/o-nKiloinlril
2-Nilro-plic»yl-a/.o-m;il'onilril
l-Nilro-plicnyl-azo-miilomtril
4-Nitro-phcuyl-azo-mnloniUil
2-Mciliyl-4-nitro-p!icnyl-a/o-malonitril
3-Mclliyl-4-nilro-phcnyl-ayo-m3lonitriJ
4-Mclhy!-2-nnro-phciiyl-a/ii-mnlonilril
2-Nilro-4-aclho\y-plicnyl-:i/o-malonitri1
2-C"lik'r-l-nilro-piK'iiyl-.izo-malonilril
3-Oilor- l-niiro-plicnyl-.i?o-malonitril
4-Clili>r-2-niiro-pliciiy1-a7o-miik)nilril
]1)icnyl-a?o-maloniiril-4-karbonsaurc-:iethyl-cster
1 -Naphtyl- jzo-malonitril
4-Uroin-I-naphtyl-azo-nialonitril
M/ I.IXS)
MI 2,5
-------
a treatment for g.i. tract worms in mono- and polygastric animals.
Fahray and Fahmy (1972) injected male mosquitos, about one day old,
with <. the LD-30 of various 4-methylaminoazobenzenes and DAB, The
males were then allowed to mate with virgin females. Results are in
Tables 130, 131, 132, 133, and 134.
TABLE ito~
GENERAL MUTAGENrC ACTIVITIES OF DAB AND ANALOGOUS SUBSTITUTED DERIVATIVES
AS INDICATED BY THE YIELD OF X-CHROMOSOME MUTATIONS: RECESSIVE LETHALS
AND VISIBLES IN ALL STAGES OF THE TESTIS
Compound!
Controls l . ,
D^B
3-Mc-DAB .
'"CIMc-DAB .
*'-ClEt-MAB '.
OEi-!VfAB C.'
«.
Dote
mM
—
10
20
10
10
20
10
10(a)
10(b)
1
5
Chromosomes
teittd
3707
2070
986
1714
1855
1509
1706
2036
1337
1614
2312
( *" >lc* injected with the adminhtrjlion vehicle only • 2'
VTnhcrorrm
>->Jic CpjrluJ li>vuc) mwtj
inti Me cmtiretJ
letruil
6(1)'
5(1)
2(1)
2
4
4(1)
10(3)
4(2)
5
13(!)
17(1)
"• (v/v) dimcthyllbrmum
in pjrenlhc^cs.
Mut alien*
visiblei
2
3
I
0
0
0
Id)
5(3)
20)
7
0
ule (DMF) in arjchu oil.
Total: per 10"
2.2 ±0.8
3,6±1.1«
1,2±0.§
2.4±0.8
6.4 ±1.9
4.7±1.2
9.4±1.5
on frequency i
2 pet 10*.
431. - ^-—.-^______
MUYAGENIC ACTIVITIES OF DAB AND ANALOGOUS SUBSTITUTED DFRtVATlVES ON Tilt
BOBBED (bb> AND MINUTE (M) LOCI
Com pound!
Controls l ...
0AB
3'-Me-DAB . . .
3'-ClMe-DAB . .
MBzO-MAB . .
/V-ClEt-MAB . .
2'-COOH-2-Mc-/V-
CIEt-MAB . .
mM
10
10
10
10
10
1
5
Gametes
observed
108202
41973
14894
21749
19629
34310
5591
6563
Ptwoofypic (&64-M)
No.
147(5) '
112(6)
39(5)
45(3)
83(1)
53(3)
W2)
10(4)
pet 10"
1.4±0.lf
2.7±0,5
2.6 ±0.4
2.1 ±0.3
4,2i0.3
1.5 ±0.2
1.6 ±0.4
Tr»i
No.
42H)
64il)
12(1)
2
iS9(l)
19
6
5
remitted />&
per I0»
0.4 ±0,06
1.5-^0.2
O.S±0.2
0.1 ±0.1
2.5 ' 0.4
0.6 ±0.1
0.9 ±0.3
S^vS.
207053
81926
28985
41658
3t677
34310
10737
12427
T,»
No.
17(2)
5
1
9
40)
1
1(1)
2
rurmtt.
rf.V
per 10'
0.08
006
0.03
0.22
0,11
O.Oi
0.13
•oo:
•00»
•OOJ
. 004
. 0.0?
.rQOJ
.:0.07
1 aid ' Stt footnote to T«bt«
Tables 130-134 reprinted with permission from Int. J. Cancer
10:194-206 (1972). Copyright by International Union Against Cancer.
188
-------
TABLE 131-
MUTAGENIC ACTIVITIES OF DAB AND ANALOGOUS SUBSTITUTED DERIVATIVES WITH RESPECT
TO THE MINUTES AND X-CHROMOSOME RECESSIVE VISIBLCS AS DETECTED BY THE
ATTACMED-X TECHNIQUE
Controls '
DAB
3'-CIMe-DAD .
jV-BzO-MAB . .
A/-CIEt-MAB . .
DOM
mM
_
20
20
10
10
Gametes
toted
55448
16739
19989
14743
17095
visible*
i
3(1)
1(1)
4
0
MffTlltfl
No.
37(11)'
9(2)
9(2)
97(5)
40(2)
per
0.7.
0 5
0.5
66
2.3
10'
, — — -
-O.I
-02
-02
•07
•04'
1 and ' S» footnote to Table 130
• MuUlion frequency in tpcrrrutocytic uagc> waj 4.3±l.4 per 10*.
TABLE >53
THE EFFECT OF THE CELL STAGE DURING SPERMATOCENESIS ON THE YIELD OF
DIFFERENT MUTATIONAL CLASSES SVITH DAD AND ANALOGOUS SUBSTITUTED DERIVATIVES
Maximal muucemc rnponte
Com pound*
DAB
3'-Me-DAB . . .
tf-BzO-MAB . .
/V-ClEt-MAB . .
2'-COOH-2-Me-M
ClEt-MAB . .
Mufational
function •
X-mutfitions
bobbed
. bobbed
X-muutions
bobbed
Minutes
X-mutations
bobbed
Minutes
X-mutations
bobbcJ
Progeny
Sampling
diys
11-21
0-3
0-3
4-6
0-3
4-6
7-10
0-3
7-10
4+;
4 + 5
Main
cell suge
Spermatogonia
Sperm
Sperm
Spermatids
Sperm
Spcrmatids
Spennatocytes
Sperm
Spermatocytes
Spermalids
Spcrmatids
Peak mutability relative to mcjn *
1.4
2.1
1.4
1.6
4.7
1.1
1.5
3.8
1.9
1.5
2.0
X
0.7
3.3 '
0.5
0.7
4.8
0.7
0.6
4.3
1.8
1.0
1.2
p
0.2
0.3
0.2
10 '
0.2
03
10"
004
02
01
1 Bjsed on /-combination te-jts fiom x1 four-fold contingency tables, where the mutjtion frequencies in the truting period"- *''•
hlKhc&i yields (derived from the most responsive cells) were compared wuh the turrcsnonding overall vjlues for the *holc r'1'**
representing the meun response of all suues of the tebtis.
TABLE >»H.
SELECTIVITY FOR THE r-RNA LOCI WITH DAB AND ANALOGOUS SUBSTITUTED DERIVATIVES AS
INDICATED BY THE RELATIVE INDUCTION OF BOBBED tbb) AND OTHER X-CHROMOSOME MUTATIONS:
RECESSIVE LETHALS AND VISIBLES; X-(/-i-r)
Compounds
D*B
*-Me-DAB
"•BiO-MAB . .
"•ClEt-MAB . .
i'-COOM-2-Me-;v-
— EI-MAB . .
Induced muutiorrj ': per 10*
X^/ + ,)
1.4±1.3
0.0
4.2±2.1
2.5 ±1.4
7.2 i 1.7
Mi
1.1 ±02
0.4 ±0.2
2.1 ±0.4
0.2 ±0.1
0.5 ±0.3
Gamete*
41 973
14894
19629
34310
12154
Induced frfr relative to other X-muutions
X« + ,)'
59
0 (3.7) >
82
86
88
w>
47
6(13.1)'
41
5
6
Ratios:
0.797-1-0.052
(3.541)
0.500 ±0.055
0.058 ±0.024
0.068 ±0.026
^ "*f induced fre>i*icnciei for the vjnoui mut.itionjl ctjis^i VKT« cjtculnlcd a» the weighted f"cjn» of t
nnicnti with the -4me compound after the *uNracrri»n al the coriesponclmi: control contributions, ba
ofthe observed value* from all
ia%cJ on Tablet I und II.
^ted on the ba^n ul the >jmplc M/C o( >crved Uir the bt>'\ and the IcuJuccJ X-mutJlutn lrequ«ncy.
r limiti of Che Ponton eM^^-tjiioin al P •• 0025.
189
-------
D. Plants
Zsolnai (1964) tested many azo compounds for toxicity to fungi
and published the results in the usual form of maximal diluted effective
dose (Table 135).
Zsolnai (1965) published some more data on the effectiveness of azo
compounds as fungistats (Table 136).
Mitra and Dighe (1968) prepared and tested as fungistats halo- and
nitrophenylazosalicyclic acids. In Table 137 Nos. 1-9 refer to R-phenyl-
azo-5-salicyclic acid, where R is 2-nitro, 3-nitro, 4-nitro, 2-chloro,
3-chloro, 4-chloro, 2,4-dichloro, 3-bromo, and 4-bromo.
E. Microorganisms
Koshimura et al (1953) reported the following tubercular-inhibiting,
maximum dilutions for substituted azobenzene: 3-methoxy-4-amino-l/40000,
2',3-dimethoxy-4-amino-l/40000, 2',3-dihydroxy-4-cyano-l/40000,
2',3-dimethoxy-4-cyano-l/40000, 2',3-dihydroxy-4-carboxy-l/160000,
2,2'-diacetoxy-l/80000, 2,2',4-trihydroxy-l/64000, 3,5,5'-tribromo-2,2'-
dihydroxy-1/20000, 3,5,5'-trichloro-2,2'-dihydroxy-l/160000, 3,5,5'-tri-
chloro-2,2'-diacetoxy-1/160000, 2-hydroxy-5-nitro-4'sulfo-, disodium
1/8000, 4-hydroxy-5-nitro-4'-sulfo-, disodium 1/20000, 4-hydroxy-4'-
sulfamy1-1/160000, 4-amino-4'-sulfamy1-1/80000, 4-diethylamino-4'-
sulfamyl-1/80000, and 2,2'-dihydroxy-l/320000.
Raynaud et al (1957) tested some azobenzenes both in vitro and
in vivo against Myobacterium tuberculosis. The following were the
only ones to show in vivo activity: 2-methyl-4-hydroxy-5-isopropyl-4'-
sulfonamido-, 2-methyl-3", 4-dihydroxy-5-isopropyl-4'-carboxy~, and
2-amino-4-hydroxy-5-carboxy-4'-sulfonamido-. Finkelstein (1961) tested
190
-------
Azo Compounds as Fungistats
Fungus
Candida albicai-.s
Cn-ptocxiccus rubt-r
Saccharom\-ces cere^iae
Trichophyton gyp^eum
Epidermophyt'in
K2ufniarT-\\ olff
Trichoihccmm ro^t_~
Peniciihuni j..\ jr.CUTI
Penic.Ilium
sirr.j.ilici!.s-irr.dri
Aspergillus elecar.b
Aspergillus nicv
Aciinc.Tiucor ri.pe-«
Boir>ns cincrea
Fusariu;n o\>sporum
im soijr.i
Phcnxl-
Ezoma'o-
rtiiril
F 2231
M -> 5 ..
v •= r>x
v: \50o
M 25.0-00
v ic»y«.
M <:\>j
M - * »'
V - ; .1
V - c.y;
.M 2 :'.>'
\( 1.-,>I
\< •> <(vi
M 2.5vj
2-TcKI-
azo-malo-
nitnl
F 2202
M •;"•"•.
\ ' * i * > ' )
M 5.000
M 25.000
\ -' ' '«-!
\ ', . f i i. '
v 5 '>>:•
M 5 OOJ
v ' ooo
•> • i uoo
'." 2.500
\; ~" <0">
\1 2 500
3-Tolyl-
azo-malo-
niln)
F/2203
M
Si
M
2 SIX)
5,000
2,500
M/25.000
h
>,•
\i
,,
N<
M
M
M
M
M
~" iK''~'
5,0-X)
5,000
' -00
2.500
2 -IX)
2,^00
2 500
2,500
j 2-Chlor-
4-To!yl- i phenyl-
a7o-ma!o-i azo-malo-
nitn! 1 nitril
F/2204 i F.7205
V,; 5,000
M/25,000
M 25 00?
M ' 1 0 CM-."'
—
M - i .OoO
M' 2 SOO
—
—
—
M
M
M
M
\(
M
M
M
M
M
M
M
M
5.003
' 5 TOO
' 2,500
.25,000
2 "* O'J".
10 f,n
2.500
2,5&J
7 SQO
' 2.500
2.500
1 2,500
•' 2,500
2.500
3-Chlor-
phenyl- |
azo-malo-|
nilnl i
F/2206 !
M,' 10.00-0 :
M/25.000 j
M/ 5,000 |
M/25.000 i
K-l -'^0 Or^0
M '2^ O')0 '
M/ 5.000
M,l 0.000
M '10 000
M; 5,000 '
M '10.00-0 '
M/ 5.000
M/ 5,000 j
M' 5.000
M/ 5,000 '
4-Chlor-
phenyl- ,
azo-malo-
nitril
F/2207 ,
M 10.000
M '50,000
M/10,000 i
M/25,000 i
M '50 000
M-25 000
M '10 000
M,'! 0.000
M 10 000
M.' * 000
M '25.000
M,IO 000
M '10,000
M '10,000 '
M/ 5,000 '
4-Brom-
phenyl-
azo-malo-
nitri!
F/220S
M 10.000
M 50,000
M 10.000
M, '25 .000
M ""* IVM
M I'J.OOO
NVS 000
M "•> 000
M' 1,000
M '25.0'X;
—
—
—
4-Jod-
phenyl-
azo-malo-
nilril
F/2209
M/ 1.000
M '25.000
M/ 5.000
M/50.000
M '50 000
M 25 oOO
—
M -25.000
M 2^ 000
M/ 5.000
—
—
—
4-Aetho\y- 2-Meihyl-
phcn>I- 4-orom-
azo-malo- phcnyl-
nilril azomalo-
niiril
F'2210 F/2211
_ M 5.000
— M 5.000
— M 5.000
M '25,000 M 25.000
M 25.000 M -
M 1
— M
_ M
^\
M 5.000 M
— \1
ODO
-------
Table 13?. Continued
Candida albicans
Cryptococcus ruber
Saccharcmyccs cerevisiae
Trichophyton gypseum
Epidermoph\ton
KfUifman-\Volff
Achonen qmnckeanum
Trichoihecium roscum
Peniciliium javanicun
Penicillium
simplicissimum
Aspergillus nheus
Aspergtllus elegans
Aspergiiius mger
Actinomucor repens
Botrytis cinerea
Fusarium oxysporum
Fusarium solani
2-Meihvl-
4-j-xl-
phen> 1-
zzo-m^Io-
i mini
i
I F2212
i
M 50.000
M 50.000
M 50.000
M 5.000
M 5.000
M 10.000
M 5.000
M 5 000
M 5.00J
M 5.000
M 2.500
M 2.500
, M 2.500
! M 5.003
N5 2.500
i M 10,000
' M 10000
M 25.000
: M 25.000
M 5.000
M 2.500
; M 2.500
: —
• M 2.500
—
—
—
—
3-MethyI-
4-brom-
phen\ 1-
azomalo-
nitnl
F 2213
M/50.000
M 50.000
M 50.000
M' 5.000
M 5.000
M 10.000
M 10.000
M 5.000
M 5.000
M 5.000
M 2.500
M' 2.500
M 2.500
M 5.000
M 1.000
M 50.000
\1 50.000
M 50.000
M 25.000
M 5.000
M 5.000
M 5,000
—
M 5.000
—
—
—
—
4-Melhyl-
; 2-brom-
' phcnyl-
azo-malo-
nitnl
F/2214
M,.25,000
M, 25.000
M;25.000
Mj 2.500
' M' 2.500
M. 10.000
M,' 5000
M 2.500
M ' 2.500
M 2,500
M' 1.000
M. 1,000
M.' 5,000
- MI 5.000
M- 5.000
M, 25.000
M 25,000
M 25.000
M 25.000
M 5,000
M' 2.500
M 2,500
M,' 1. 000
M 2,500
M, 1,000
(M, 1.000)
2-Erom-
4-acthoxy-
phenyl-
azo-malo-
nitnl
F/2215
M/25,000
M/25,000
M/25,000
M/ 1 000
M/ 1,000
Ml 5000
Ml 2,500
M' 1.000
M' 1.000
M/ 1,000
—
—
—
M/50,000
M; 50,000
M '50.000
M/50,000
—
—
—
—
—
(M/ 1,000)
(Ml 1,000)
(Ml 1.000)
--
2,5-Di-
chlor-
phenyl-
azo-nialo-
nitril
F/2216
M/50,000
M, 50,000
M '50,000
M' 5.000
M' 5.000
M 10.000
M- 5.000
M; 5.000
M 5.000
M 5.000
M 2.500
M, 2.500
M 5.000
M 10.000
M 5.000
M 50.000
M 50.000
M 50.000
M 50.000
M 5.000
M 10.000
M 2.500
M 1,000
.•» IU.UVJ
M 2.500
M 1.000
M 1.000
M 2.500
I 3,5-Di-
1 brora-
1 phenjl-
! azomalo-
] nitril
: F./22I7
M/50,000
M' 50,000
M/50,000
M, 25.000
M; 10.000
M 25.000
M 10.000
M' 5.000
M 5.000
M- 5.000
M 2.500
Mf 2,500
_
M 10.000
M, 1,000
M 50.000
M 50,000
M 50.000
M 50.000
M 10.000
M. 10.000
M' 10,000
M,' 1,000
.•Vl. .),WU
—
—
—
—
2-Chlor-
4-brom-
phenyl-
azo-malo-
nitnl
F/2218
M/50,000
M '50,000
M/50,000
M/10,000
M' 5.000
M/25.000
M 10,000
M' 5.000
M 5.000
M, 10.000
M ' 2 -00
M; 2.-: 00
M/10,000
M; 10. 000
M/ 10,100
M/50.000
M 50,000
M 50.000
M 2^.000
M 10.000
M' 1 0.000
M 10,000
M; 2.500
1VH ^,UUU
M' 2,500
M' 1.000
M/ 1,000
M 1.000
3-Chlor-
4-brotn
phem 1-
azomalo-
nilnl
F/2219
M/50,000
M 50.000
M; 50.000
M/10.000
M' 5.000
M 25.000
M 10.000
M 5.000
M 5.000
M 5 000
M 2.5CO
M, 2.500
M 5.000
M 10.000
M 10.000
M 50.000
M 50.000
M 50000
M 25.000
M '.O.OOC
M 10000
M 10.000
M 2.500
IM IU.UUU
—
—
2-Methyl-
' 4.6-di-
brom-
. phen\I-
! azo-nialo-
nJtnl
1 F 2220
i
j M/50,000
i
M, 50.000
' M/50.000
, Mi 2.500
M' 2.500
M 10.000
M 5.000
M 2 500
M 2 500
M 2.500
M 2.500
M 2500
500
•- ". 000
M 2 500
M 25.000
M 25.000
M 25 000
M 25 000
M 5.000
M 5 000
\: 5.000
M 1.000
>1 ^.l/JU
M 1.000
M 1.000
M i.OOO
\1 1.000
2-Chlor-
4.6-Ji-
broin-
phen>l-
azo-malo-
nitnl
F/2221
M/50,003
M, 50.00-0
M 50,000
M' 2,500
M 1.000
M 10.000
M 2,500
M 1.000
M I.O'JO
M 1.000
M 1.000
M 1,000
M. 2,500
M 10.000
M 2.500
M 25.000
M 25.000
M 25.000
M 25.0- X)
\1 5.000
M 5 000
M 2.500
M 1.000
ni ^.uuu
M 5.000
M 2.500
M 2.500
M 5.000
' 3-Chlor-
' 4.6-di-
brorn-
; phen>i-
azo-malo-
ni'ril
F '2222
M 50000
M 50.000
M.50.0CO
M 5000
M 2.500
M 10.000
M 2.500
M 2.500
M l.tXO
M :.?oo
M l.»j
M l.OCO
\: 5.0CO
\! 5.0-IO
M 5,C'.'O
\1 50.0X)
\i 5oc-:o
M 50 'XO
\1 25.1VO
\1 10.000
\1 10.000
M 5.WO
M 1.000
M 2.UUU
—
-------
Table 13$. Continued -
4-Chlor- 2,4,6- ! 2-Nitro-
• 2-6-<3i- Tnbrom- phenyl-
brom- pheml- , azo-malo-
pMiyl- azomalo ' nitril
azo-malo- nunl
nitril
F.2223 F/2224 . F/2225
3-Nitro-
phenyl-
azo-malo-
nitnl
4-Nitro- 2-Meihyl- 3-Meihyl- [ 4-Meth>I-
phenyl- 4-nitro- ' 4-nuro- ' 2-rr,tro-
azo-mala- phcnyl- ' phenyl- i phen>l-
niinl
i a2o-malo- azo-malo- i azo-rrwlc
njinl i nitril ' nitril
F/2226 F.-2227 F/2228
F 2230
2-Nitro- ' 2-Chlor- | 3-Chlor-
4-ae;ho\>- 4-nitro- > -i-nuro-
phenjl- phen>l- | phen\l-
azo-malo- azo-malo- j izo-rnalo-
nitril nitril I nitril
F 2231
F 2232
F 2233
M 50,000 M 50.000 , M/10,000
M 50.000 M 50.000 M/10,000
M/10,000 M/10,000 My 10.000 i M'10,000 M, 10,000
' ! i
M/10,000 ! M/10,000 ; M/10,000 M/10,000 j M 10.000
M' 10.000 M 10.000 ! M 50.000
I
M "10,000 M 10.000 i M 50,000
Candida albicatts |
Cryptococcus nlber j
Saocharomyccs cercvisiae ;
Trichophyton gypsum j
Epidermophyioh |
Kaufman-Wolff
Achorion quinckeanura ,
Trichothecium roseum
PentciHmm jav^nicum I
Penicillium <
simplicissimum '
Aspergillus nixeus
Aspergillus elegans
Aspergillus niger
Actinomucor repcns
Boiry'.is cmerea
Fusanum ox><;porum
Fusar mm solani
^1 .7V,m.V
M 2,500
M 2.500
M 5.000
M 2.500
M 1,000
M 1,000
M 2.500
M" 1.000
M; LOGO
M' 2.500
M 5,000
Nf 2,500
M 25,000
M 25.000
M 25iOOO
M 75,000
M 2,500
M 2,500
M 2.500
M 1,000
M 5.000
M 2.500
M 1.000
M 1.000
M 1.000
• ' -"•""«
M 2.<00
M 2.500 ,
M 5.000
M 2.500 .
M 1.000
M; i.ooo :
M1 1.000 I
M 1.000
'" ' roo ;
1
V l.-OO
V ,0000
M : -oo
M -15 .GOO t
j
M 25 000
M 25 000
M 25 000 i
M 5.000 ;
M- 2.500
M 2.500 '
M 1.000'
M 5 000
M 1 .000
M 1 .CAT
M 1 .000
M 1.000
>l; iv,uvu
Ml 5,000
M' 5,000
M 25,000
iviy 1 U,VAAJ
Ml 5,000
M' 2,500
M/10,000
M, 10,000 1 Ml 2,500
M,' 5.000 Ml 2,500
Ml 5,000
Ml 2,500
Ml 5,000 M/ 2,500
— i —
— J —
1
Ml 1,000 —
M; 5,000 MI 1,000
M' 1,000 —
M/25,000
M/10,000
M '25,000 M' 10,000
M '25.000 - M/10,000
M, 25,000 : M/;0,OCO
M/ 5.000
M/ 5,000 —
— M/ 1,000
M' 1,000 i —
M 2,500 —
M,' 2,500 —
M/ 2,500 —
l\l; 1U.XAAJ
M.' 5.000
M' 2.500
M 10.000
MI 2.500
M' 1,000
M/ 1,000
Ml 1,000
—
—
—
M/ 1,000
M; 5.000
M' 10.000
M; 5,000
M; 10,000
M' 1.000
Ml 1.000
M' 1,000
M/ 1,000
—
Ml 1,000
M/ 1.000
IM, JU.IAAJ !
,
Ml 5.000 '
M/ 1,000
— ;
i
1
—
—
M' 2,500 '
Mi 2,500 '
M/ 2.500
M 25,000
M '25,000
M 25.000
M 10.000
M; 1,000
M' 2,500
M1 1,000
M 1.000
M/ 1,000 -
—
M; i.ooo
M 2.500
M/ 2.500
IVI; IU.UUV
M/ 1.000
M/ 1,000
M' 5.000
M/ 1,000
—
—
—
—
—
M/ 1,000
M' 1,000
M/ 1.000
M '25,000
M 50,000
M. '50.000
M; 10,000
M1 1,000
M' 1.000
M ' 1 ,000
M; i.ooo
M; 1,000
—
—
(M,!OQO)
(M.IOOO)
j IM, iij.uw :
' M' 2.500
; M 2.500
M 5.000
M 2.500
. M' 2.500
M 2.500
M 1,000
—
—
M 2.500
M 5.000
M 2.500
M 50.000
1
M 50 000
M 50000
M 2-000
\1.. 2.500
M 2,500
M' 2.500
—
M 1.000
M. i.ooo
!\lt 1U.UVU
M' 1.000
Mr 1.000
M 2,500 •
M 1.000
M' 1.000
M' 1,000
M ' 1 .000 '
—
— i
i
__
M 25,000
;
M 50.000
M 50.000
\1 25.000 .
M. 1,000 '
M, 1.000
— I
—
—
—
;vi i V.UVA; ;
j
M; 2.500 •
M ' 2,500 '
M 1,000 .
—
— i
i
1
i
M 2,500 '
M 2,500
M' 2,500
M 50,000
i
M 50000
M 50.000
M 25,000
M 1,000
M 2.500
M 2,500
M 2.500
M' 2.500
(M iOOO)
M 1.000
\1 1 ,000
M- I.OOO •
-M _-'.VJW
M. 5.000
M 2.500
\1 10.0M
M 2.5CO
M 2.503
M 2,5^0
M 2.500
M LOCO
M 1 ,000
M 1 000
NT : 5,-J
M 1 COO
M 50.000
M 50 0>X>
M 50.LT-3
M 25.000
M. I.OOO
M 2.500
M 1.000
— .
\r i.ooo
M 1.000
M 1.000
-------
Table 135- Continued
Phen> I-
azo-acel-
essig-e>ter
F/2271
3-TolyI- i
. azo-acct- ;
'. essig-ester '
i
i '
\ F/2272 |
4-Toi> 1-
azo-acet-
essig-ester
F/2273
J 3-ChIor-
| phen>I-
' azo-acel-
; essig-esler
i
i F/2274
4-Chlor-
plienyl-
azo-acet-
eisig-esier
F/2275
' Phen\ 1-
azo-m^lon-
i saurc
1 diaeth>I-
1 ester
j F/2276
: 4-ToiyI-
i azo-malon-
saiirc-
diacth\l-
'• esier
| F/2277
4-Chfor-
phen\l-azo-
malonsaure
diacth>l-
ester
F2278
!
Candida albicaris
Cn-plococcus ruber
; _ _ _ • _ — (M ioco)
Tnchonh> ion gipseum
Epidermophyion KaufnanAVolff
Achorion qumckeanuni
Trichoihecium roseum
Pimicil hum ja\ anicum
Penicilbum simpiicissinium
-\spergillus ni\eus
-\spergillus elecans
\sperg:llus niger
\c;inorriL:cor repens
Botrytis cinerea
fusarium ox>sporoiri
Fu^ariuni solani
A (-) means that a concentration of M/1000 had no effect
(M/1000), but not M/1000, means that this concentration had a partial effect
Reprinted with permission from Biochem.
Pharmacol. 13:285-318 (1964). Copyright
by Pergamon Press Ltd.
M i.ooo
M, 10.000 i
M/1 0.000 :
M/ 10.000 ;
M; 5.000 !
— •
—
—
M' 1.000
(M 1000) •
(M 1 000) i
M 1.000 ,
M/1 0,000 '
M/1 0,000
Ml 1 0.000 '
M/ 5,000
—
— ,'
—
— .
—
—
—
M, 10.000 ,
M/IO.OOO :
M 10.000
M 10.000
(M 1000)
M-1 1.000
(M 1000)
—
—
—
—
M 10.000
M' 10,000
M' 10.000
M 10.000
CM, 1000)
M, 1.000
fM/1000)
—
—
—
—
M
M
M
M'
M;
M/
(M
M'
M
\*
ki1
10,000
10,000
10.000
10,000 :
1,000
1,000
,1000)
1,000
1,000
!,000
1.000
M
M
M'
M
2.500 ,
2.500 j
2.500 1
1.000 j
i
—
—
—
—
—
—
M/
M'
M/
M.'
2,500
2,500 ,
2.500
I.OOO
—
—
—
—
M
M
M
M
5,000
5.000
5.000
2.500
—
—
—
—
-------
Table 135. Continued
4-CMor-
phenyl-
azo-c% an-
essig-
saure-
acthyl-
e^ier
F 2253
_ —
—
—
Phenyl-
4-Chlor- ! Phenyl-
azo-cyan- ! phenyl-
azo-cyan-
essig- : azo-cyan- essig-
saure-
essig-
anilid saure-
anilid
saure-
(4'-chlor-
anilid)
t \
F/2257
F/2259 F/2260
—
— —
— , —
— —
—
4-Chlor-
phenyl-
azo-cyan-
essig-
saure-
(4"-chlor-
anilid)
F/2262
—
—
— «
; Phenyl-
azo-cyan-
', essig-
| saure-
; hydrazid
i
! F/2263
I
—
—
—
1 4-Tolyl- j 4-ChIor-
' azo-cyan- ( phenyl-
; essig-
aio-cyan-
saurc- essig-
| hydrazid i saure-
: I hydrazid
i F/2264 F/2265 ,
i i
—
—
~—
M/ 5,000 •
M/ 5,000
M/ 5,000 •
Pheml-
az?-
acaui-
aceton
F 2266
—
—
__
3-To!yl-
azc-
a^--e;\ I-
-------
Table 13$. Continued
CandiCa albicar.s
Cr\p!i.»c<:>ccus ruber
Saccharomyces cere\isiae
TnchcrbMon gypseum
Hpiderr.ioplnion
Kaufmai)-\\ olff
Kchorion qumckeanum
rrichu:hecium roseum
I'enic'llmm ja\anicum
Pencil. em
simplicissimunt
\sperg:l!us nheus
V=pereillus elegans
\.sperg.'llus nicer
Actmomucor rcpens
Botrytii cinerea
Fusarium oxssporum
f-usarium solani
4_
;-
p.
>.',
M
M
M
M
M
\i
M
M
M
M
M
M
_ f| •„•.!--
rr:i\>-
icn>l-
-rrulo-
•.;:nl
-i -i ^ •
5l'l,-Hj
50 OuO
50 OXi
5.000
2500
25.000
5.000
5.000
2.500
2.500
—
2.5CO
5.0CO
2.500
Njona
50000
50.000
25.000
2.500
5000
l.OCO
! .0:0
"* ^no
KOOQ
2.500
2.500
2.500
Pi
ii/_
PI
ka
s
at
t
F
M
\i
M
M
M
M
M
M
M
M
M
M
M
M
M
,or,x |.
-rri,-. 1."
.rJ-4-
rbon-
,'jrc-
•ih}I-
-•icr
2238
25.fKX)
25.000
25.CKK)
1.000
1.000
f.f.KW
2.500
2.500
2.500
2.500
—
—
5.000
—
2; 000
25000
25.000
25.000
—
—
—
—
—
—
—
—
--
).>,
2/
;
*'i
M
M
M
M
M
M
M
M
M
\]
M
M
M
M
M
M
M
M
M
M
M
M
M
V-,T-
1-1)
nil
- '2.
51
50
50
5
. 2
10
>
1
T
">
1
1
1
' S
1
10
|-,v|.
l.'iO-
-il
}
:^
.0(10
.000
.000
000
.SCO
000
.000
.500
.500 '
.500
000
,000
.000
.000
000
.(100
'25.000
25
T-,
000
.000
5.000
>
1
1
!
000
000
000
QfiQ
1
4-fiiom-
-1-n.ipli-
t} l-.l/D-
malo-
nitril
F/2244
M- 50,000 !
M/50,000 |
M, 50,000 ;
I
M/ 5,000 >
M/ 2/00 ;
M, 10,000
M 5,000
M/ 2,500 i
M/ 2,500 ,
M ' < 000
M' 1.000
M' 1.000
M, 1,000
M. 2,500
M, 1,000
M' 10 000
M '25.000 '
M '25,000 '
M/25,000 '
V, 5,000
M,' 5,000
M/ 2,500
M; 1,000
M/ 2,500
—
—
—
—
A/.ohcn- i 3.' -Di-
, . i , ,
7^1-4-nzo- melml-
malo- Jiplicnyl-
nitn) cn-4.4"-
bi>-(azo-
; malo-
nunl
F 2245 i K-2247
i
M/50,000 : M 10,000
M, 50.000 ' M/10,000
M/50,000 ] M' 10,000
— i —
— 1 —
— ' —
Phen> I- | 4-Tol>I- : 4-Ciilnr- ! Phcn>!-
azo-cv,in- azo-c>an- ! phenyl- ' a/o-c>^n-
cssig- I essig- azu-cyan- ; e>->ig-
saure : saure essi;> i saurc-
meth>l- ' methyl- Sdurc- aeihyl
ester ' ester meihyl- ester
I ester
F/2248 I F/2249 F/2250 F 2251
— . — i — —
— , _ _ , _
i i '
i
1 —
— ' — -) — -_
— , — — —
— ' — i — — — —
— — .
— . —
— - —
— —
— —
— —
— —
Ml 2,500 —
M/ 5,000 —
M/ 5,000 —
i
— — 1 — _
— ' — — —
— — t — _
— — ; — —
— — _ __
M/ 5,000 , M, 10,000 • M/25,000 M 10000
1
M/10,000 '< M/10,000 M/25,000 M 10.000
M/10,000 ' M, 10.000 M;'25,000 M 10000
M/ 5,000 — i Ml 2,500 M/ 5,000 .' M'10,000 M 5000
t
— — — - , — M : 500
— — — ; — ! — M 1.000
— —
— — — M 1.000
— — — ; — —
— : — — i — ' . — —
. — — — j — — .
_ — — _ —
j.-j (.,|v|_
azo-oan-
el-
esier
F. 2252
—
—
— -
—
—
—
—
—
—
—
—
M 10 COO
M 10.000
M 10.000
M 5 000
—
—
—
- —
—
—
—
-------
Table "136. Azo Compounds as Pungistata
Fungus
Candida albicuns
Cryplococcus ruber
Saeeharomyces ccrcvisiae
Tnchuphyton gypseum
Epidortnophyton Kauf-
nwii-Wu!I)
Aclioru >n quinckciinum
'I'ricliuilicciuin rovcum
PcniciIIium javanicum
I'enic!ilium Minplieissi
Aspergillui nivcus
Aspcr^illns elegans
Aspcrgillus nigi-'r
Actin«>mucor rcpens
Botrytii, cmcrca
Fuiitnum oxysporutn
Fusurmm sohmi
4-Acetyl-
phenyl-azo-
malonitril
F/2313 ..
M/10,000
M/10,000
Mi 5,000 ,
Ml 5,000
M/ 5,000
M/10,000
Ml 5,000
M/ 1,000
M/ 1,000
M/ 1,000
M/ 2,500
M/ 2,500
M/ 2,500
M/ 2,500
Ml 2,500
4-Acetyl-
phenyl-azo-
malonitril-
semicarbazon
F/2318
M/ 2,500
Ml 2,500
M/ 2,500
M/ 2,500
M/ 1,000 '
M/ 2,500
M/ 1,000
M/ 1,000
—
~~~
M/ 2,500
M/ 2,500
Ml 2,500
M/ 2,500
M/ 2,500
4-Acctyj-
phcnyl-azo-
malonitril-
oxym
F/2321
M/ 1000
M/ 1,000
(M/1000)
M/ 1,000
—
—
—
—
_
— ~ •
i
—
_
,
—
—
4-AcetyI-
phcnyl-azo-
rruiloniiril-
phenyl-
hycirazon
F/2322
M/10,000
M/10,000
Ml 5,000
M/ 5,000
Ml 2,500
M/ 5,000
M/ 1,000
M/ 1,000
M/ 1,000
Ml 1,000
M/ 2,500
M/ 5,000
> Ml 5,000
M/ 5,000
M/ 2,500
4-Acetyl-
phenyl-azo-
maionilril-
salizyioyl-
hydrazon v>
F/2323
Ml 5,000 .
M/ 5,000
M/ 5,000
M/ 2,500
M/ 2,500
Ml 2,500
—
—
—
^ „
_
"x —
_
"__
4-Acetyl-
phenyl-azo-
malonitril-
isonikotinoyl-
hydrazon x
F/2324
. . M/ 2,500
Ml 2,500
(M/1000)
Ml 1,000
—
—
—
—
—
Ml 2,500
M/ 2,500
M/ 2.5CO
Ml 2,500
M/ 2,500
4-Acety!-
phcnyl-azo-
cyancssigsiiure-
methyl-ester-
arnino-
guantclon
hydrochlorid
F/2328
M/ 2,500
Ml 2,500
Ml 1,000
M/ 1,000
—
—
—
—
—
— -
Ml 1,000
M/ 2,500
M/ 2,:OP
M/ 2,iOO
4-(4'-Acctyi-
phenyl-azo)-
3,5-dimethyl-^
pyrazolyl-
1-amidin-
aminoguanidon
dihydrochlorid
F/2330
M/10,000
M/10,000
M/10,000
—
—
—
—
—
, —
—
Z^,
—
—
—
—
Reprinted with permission from Biochem
Pharmacol. 14:1325-1362 (1965).
Copyright by Pergamon Press Ltd.
-------
Table 136. Continued
•o
CO
Candida ulbicans
Cryptocoixus ruber
Saccharomyces ccrevisiae
Trithnphyion gyp^eum
Epidcrmophylon ICauf-
nun-WoItr
Achoriun quinck/Mnum
Trichothecium nr>cum
Pcnicillium javanicum
Pcniullium Mmplicissimurn
Aspcrgillus nivcus
4-(4'-Acety|-
phenyl-azo)-
3-methyI-
pyrazolon(5>
yl-1-amidin-
ainino-
guanidon
dihydrochlorid
F/2331
M/ 5,000
M/ 5,000
M/ 2,500
—
—
—
—
—
—
—
_
—
4-Phenyl-azo-
3,5-dimethyl-
pyrazolyl-1-
amidin
hydrochlorid
F/2344
M/ 5,000
M/ 2,500
M/ 5,000
M/ 2,500
• M/ 1,000
M/ 1,000
M/ 1,000
M/ 1,000
(M/1000)
M/ 5,000
M/ 5,000
Ml 5,000
M/ 1,000
4-{3'-Tolyl-
azo)-3,5-
dimethyl-
pyrazolyl-1-
amidin
hydrochlorid
F/2345
M/ 5.000
M/ 5,000
M/ 5,000
M/ 2,500
M/ 1,000
M/ 1,000
M/ 1,000
M/ 1,000
(M/1000)
M/ 5.000
M/ 5,000
M/ 5,000
M/ J.OOO
4-(4'-Tolyl-
azo)-3,5-
dimethyl-
pyrazolyl-l-
amidin
hydrochlorid
F/2346
M/10,000
M/10,000
M/10,000
M/ 2,500
M/ 1,000
M/ 1,000
M/ 1,000
M/ 1,000
(M/1000)
M/ 5,000
M/ 5,000
M/ 5,000
M/ l.OCO
4-(3'-Chlor-
phcnyl-azo)-
3,5-dimcthyl-
pyrazolyl-1-
amidin
hydrochlorid
F/2347
M/10,000
M/10000
M/10,000
M/ 2,500
(M/1000)
(M/1000)
(M/1000)
—
(M/1000)
M/10,000
M/10,000
M/10,000
M/ 2,500
4-(4'-Chlor-
phenyl-azo)-
3,5-dimethyl-
pyrazolyl-1-
amidin
hydrochlorid
F/2348
M/10,000
M/10,000
M/10,000
M/ 2,500
M/ 1,000
M/ 1,000
M/ 1,000
M/ 1,000
(M/1000)
M/10,000
M/10,000
M/10,000
M/ 2,500
4-(4'-Tolyl-
azo)-3,5-
dimethyl-
pyrazolyl-1-
thiocarborj-
siiureamid
F/2356
M/ 2,500
M/ 2,500
M/ 2,500
—
—
—
—
—
—
M/ 5.000
M/ 5,000
M/ 5,000
M/ 5,000
4-(3'-CWor-
phenyl-azoV
3,5-dimethyl-
pyrazolyl-1-
thiocarbon-
saureamid
F/2357
,r; M/ 2,500
M/ 2,500
M/ 2,500
—
—
—
—
—
—
Ml 5,000
M/ 5,000
M/ 5,000
M/ 5,000
A->pcrt;illir,
Aumoniucor repcns
Hutryli-, cincrca
1-uiarium o.xysp
I-'u>>ariuin solani
-------
Table 136. Continued
4-<4'-Chlor-
plu-nyl-azo)-
3,5-dimcthyl-
pyrarolyl-
thmcarbon-
siiurcamid
F/2358
Candida stlbicans
Cryptoeoecus rubcr
Satehartmiyces ccrevisiae
.Trieliopliyton gypseum
Ephlermuphyton Kaufman-Wolff
Achurion i|trinckcanum
TrithotlKcium roscum
PcniciUnini .uivumcurn
Pemcillium simplicisbimum
A->pcri;i]lu< nivcus
Aspcrj'illyv clcguns
A->p<-'i|',illus nif.cr
Actinoinncoi' rcpcns
Holrytis uiicriM
i'liNaiuniv iixyiporum
l:us:uiuin sulani
M/ 5,000
Ml 5,000
Mf 5,000
Ml 5,000
TABLE 137.
Antlfungal Activity of Salicylic Acid Derivatives
No.
1
2
3
4
5
6
7
8
9
Minimum inhibitory
Candida species
albicaftS | krusei
0.01 0.01
(11.8) (12.0)
— —
0.25 0.01
(11.5) (11.8)
0.01 0.01
(13.2) (U.O)
— 0.01
(15.5)
0.1 0.01
(10.5) (10.5)
0.1 (0.1
(13.5) (15.9)
0.1 0.01
(15.5) (10.0)
0.1 0.1
(11.1) (11.0)
Note— 1.
concentration
for pathogenic fungi (Percent. W/V)
Triehophytoa ipecies
verrucosum j
0.1
(12.5) .
—
—
0.01
(25.0)
__
1 _ _ '
~.
,
-_
—
mentagrophytcs
0.1
(15.1)
— ;
, 0.01
(20.5)
0.01
(18.5)
—
0.01
022.5)
*.
0.01
(24.5)
0.01
(25.8)
rubrum ] tonsurans
0.01 0.01
(16.1) (18.3)
— —
0.01 —
(15.9)
0.01 0.01
(20.0) (19.9)
— . - • — \ x
— ' _ ''
0.1 —
(13.0)
0.25 —
(18.9)
— —
Microsporum
ipecies
canis | audouini
0.01 0.01
(20.0) (19.5)
— —
0.01 —
(17.8)
0.1 0.25
(15.6) (20.1)
_-. .M
. __ .- ___ *
5 '
_. — _
„ . i r ^^
0.1 —
(36.2)
Epidcrmo-
phyton
floccosum
0.1
(15.4)
....---.,
0.25
(18.1)
.,,
•
n -
t
'._ ..• l. ' ,
S.
—
Figures in brackets indicate diameters of zones'of inhibition in mm.
2, ( —) indicates no activity.
199
-------
Trypan Blue and other dyes/stains for protection against Newcastle
disease virus (NDV) in chick embryos. An effective dose, 1 mg, of
Trypan Blue was much less so if given after the virus injection instead
of before. The effectiveness of two dosages of Trypan Blue are given
in Table 138, also (in Column A) an indication that a preventive dose
would have to be adjusted to the amount of infection expected.
TABLE 13 8.
Protective effect of Trypan blue agaimt NDV in chick embryos
, — _—
Loj V'uui Dilution
-10
-9
-8
-7
-6
-5
ItorUlity*
4/10
18/20
39/40
Tiyptn blue'
A
2/20
6/19
17/20
19/19
B
20/20
c
4/40
D
40/40
•s.
4/20
F
20/20
• No. dead/total, 92 hr after inoculation (data combined from two experiments).
»A, Trypan blue, lot 11003, 1000 jig/egg, immediately prior to virus; B, Trypau blue, lot 11003, 100
ff/egg, immediately prior to virus; C, Trypan blue, lot 16124,1000-^g/egg, immediately prior to virus;
D, Trypan blue, lot 16124,100^g/egg, immediately prior to virua; E, Trypan blue, lot 16124, 1000 ^g/
tgj, 24 br prior to virus; F, Trypan blue, lot 16124, lOOO^g/egg, 24 hr after virus.
Reprinted with permission from J. Immunol
87:707-13 (1961). Copyright by Williams '
& Wilkins Company.
Table 139 presents the results of the other compounds tested, all in 1
mg doses.
Zsolnai (1964) tested many azo compounds against a variety of bac-
teria, with results in Tables 140 and 141, in units of minimum molar
dilution still effective.
Zsolnai (1965) reported the results of some more azo compounds, in
Tables 142 and 143.
200
-------
COMPOUND
TA1JL.K 131-
Effect of Trypan blue and other compound* on Newcastle disease virus in chick embryos and on .hetnagglutination
STRUCTURAL FORMULA
morccTivi*
HCUMQV.UT1MM ION
COMPOUND
STRUCTURAL FORMULA
"WTtCTivf*
ro
O
Tr»M
Ci.t govVN •N -/~V-^"V-" • "Vv1*!
JL IJ i=/>—t L il J
•^^^^ CM, CM. •^*!^*^S«.«I.
M.SOj SOjNotM» CM» NoSO, Wj *
bran V
Chronoliop* ZK
FMI dmun CH
Bxtflck (Corlil
•CH,
H,N OH
OH NH,
SOjNo
OH
OCH,
' N_(MV_^<1
•SOjNd
NHj
NoSO, NoSO,
NHj OH
-N • H_jT\^\JI • N_
cfr
NoSO,
-NH,
OH OH
• N.
NoSOj
OH NH-COCH,
NoSOj SO,Na
N • N
No 30,
Fill GfMn FCF
M*lhyl Oran««
Mtlhyl nd
PMICMU 211
MM
CM.CM,
C«H, •
OCH,
-N-ON«
"•SO,
.COOH
N-N j**
VI."
CH,
'SCjNo
NO,
*IOOO >A4 «(
admmlttticd allantolcalty ptlor to
Reprinted with permission from J. Immunol. 87:707-13 (1961). Copyright by Williams & Wilklns Co.
-------
table 1UO, Azo Compounds as Bacterlostats
s
Ph«nyi- 2-Tclyl- 3-Tolyl-
szo-malo- azo-malo- aio-malo-
BacfceriUITl nitril mini nilril
F-2201 f 2202 F/2203
Staphylococeus aureus
Duncan M 10.000 M, 10,000 . M/25,000
Staphylococcus aureus
p%ogenes M 10.000 M 10,000 M/10,000
Stapbylococeus albus M' 10,000 M 10.000 M/ 10,000
Shieella dysenteria*
Flexner M 5.000 M- 2.500 ' M/ 2,500
ShicciladvscnteriacSonne M' 5.000 ' M' 2,500 I M,' 2,500
Salmonella t\phi M 10.000 M 10.000 j M/10,000
Salmonella paratvphi M 5.000 ' M 10,000 ; M/10,000
Eschenchiacolicomtnunii M 2.500 • M' 1.000 • M/ 1,000
Acrobacter aerogenes '• M 2.500 M' 1,000 i M/ 1,000
Proieus vulgaris . M' 2.500 ' Ml 1.000 ' M/ 1,000
Pseudomonas pyocyanea ! — 1 — ' —
Pseudomona* fluoresoens ' — ' — —
I : i
• 2-M«hyl- 3-Methyl- 4-Methyl-
! 4-j"xl- 4-brijm- • 2-brom-
' phsnyl- pheml- j phenyl-
| azo-ituio azomalO" azo-malo-
j nitnl nitril niiril
i
! F2212 F2213 F/2214
i
Staplivlocoocus aureus i
Duncan • ; M 50.000 M/50.000 . M/25,000
Slaphflococcus aurjus !
pyo'gcnes JM50000 M 50.000 M.25.000
Siaphslococcusalhus ' M 50.000 M 50.0*3 M'25.000
Shicflla dysenteriae
Flexner ! M 5.OM M1 5.000 M; 2.500
Sliigclla dvsemeriae Sonne M 5.000 M 5.000 • M' 2.500
Salmcroi-11.1 urhi M !0.m.O M 10.000 M 10.000
Salmonella p'aratvphi M l&JO M 10.000 M,' 5.000
EscIierichiacoHcommunis N5 5.KO M 5.000 M1 2.500
Aerobactcr aerogenes • M 5.00>J M 5.000 M' 2,500
Proteus vuleans . M 5.COO M 5.000 M 2,500
Pseudomonas pvocvanea ; M 2.500 M 2.500 M' 1.000
Pseudomonas fluore«ens ' M 2.500 M 2.500 M-' 1,000
4-Tolyl-
azo-malo-
nitril
F/22M
M/10,000
M/10,000
M/10,000
M/ 5,000
M/ 2,500
M/10,000
M/ 5,000
Ml 2,500
M/ 2,500
M/ 2,500
2-Brom-
4-aetho,xy-
phenyl-
azo-malo-
nitril
F/2215
M/25,000
M ('25,000
M/25,000
Mi 1.000
Ml 1,000
M/ 5000
M/ 2,500
M' 1.000
M' 1.000
M/ 1,000
—
_
2-Chlor-
phenyl-
azo-malo-
nitril
F/2205
M/25,000
M/25,000
M/25,000
M.' 10,000
M/10,000
M, 25,000
1 M/10.000
M ' 2.500
Ml 5,000
! M,' 5,000
i Ml 1,000
i M: i.ooo
2,5-Di-
chlor-
phcnyl-
azo-malo-
nitril
. F/2216
M/50,000
1 M 50.000
' M '50,000
M • ,00(1
- M .000
M 1 .000
. M .030
' M- .000
, M .000
M .000
M .500
' M ,500
3-ChIor-
phenyl-
azo-malo-
nitril
F/2206
M/50,000
M/50,000
M/50,000
M/10,000
M/10,000
M/50,000
M/10,000
M/10,000
M/10,000
M/10,000
M/ 2,500
Ml 2,500
3.5-Di-
brom-
phcnj'l-
azo-malo-
nitril
F/2217
M/50,000
M .'0,000
M, 50,000
M:\\000
M; 10.000
M -25.000
M 10.000
M' 5.000
M 5.000
M 5.000
M 2,500
M/ 2,500
4-Chlor-
phenyl-
azo-malo-
nitril
F/2207
M/50,000
M/50.000
M/50,000
M/10,000
M/10,000
M/50,000
M/10.000
M/10,000
M/10.000
M/10.000
Ml 2,500
M/ 2.500
2-ChIor-
4-brom-
phenyl-
azo-miilo-
nitnl
F/2218
M/50,000
M '50,000
M/50,000
M/10,000
M' 5.000
M. '25.000
M 10,000
M 5.000
M 5.000
M 10,000
M' 2,500
M; 2,500
4-Brom-
phenyl-
azo-malo-
nilri!
F/2208
M/50,000
M/50,000
M/50,000
M '10,000
M/10,000
M -'25,000
M ,'25,000
M/10.000
M.1 10.000
M/10.000
Ml 2,500
Ml 2,500
3-Chlor-
4-brorn
phen> 1-
azomalo-
nitril
F/2219
M/50,000
M. 50.000
M;50.000
M 10.000
M1 5.000
M 25.000
M 10.000
M 5.000
M 5,000
M 5000
M 2.5CO
M. 2.500
4-Jod-
phenj!-
aio-mJiIo
nitril
F/2209
M/50,000
M/50.000
M/50,000
M/' 10 000
M/10.000
M '25.000
M'10.000
M; 10.000
M '10.000
M 10.000
Ml 2.500
M/ 2.500
2-MethyI-
4.6-dt-
brom-
phen\l-
azo-malo-
niinl
F2220
M/50,000
M, 50.000
M.'SO.OOO
M/ "> ^00
M' 2.500
M 10.000
M' 5.000
M 2.500
M 2.500
M 2.500
M 2.500
M 2 500
4-Aeihoxv- 2-Meihyl-
phenyl- 4-orom-
" azl- . phenji-
azo-malo- azo-maio-
nitnl pitril
F;2221 F'2222
M/50,000 M 50.0UQ
M/50.000 M 50.000
M 50,000 M. 50,000
M 2 ''OO \i < OX)
M 1,000 M 2 ;rvt
Si 10,000 M 10.000
M 2,500 M 2.:00
NI i.fK» M :.f»
M 1.000 V! ] C-X-
M !.0;>0 \1 2.-VO
\'. 1000 M 1..-/J
• ! 000 M 1.000
Table reprinted with permission from Biochem. Pharmacol. 13:285-318 (1964). Copyright by Pergamon
Press, Inc.
-------
fable 1IiO. Continued
1
I
i
—
Siaphyiococcus aureus
Duncan
Staphylococcus aureus
pyogcnes
Staphylococcus albus
Shigella dysenteriae
Flcxner
Shigeila dysenCeriae Seane,
Salmonella typhi
Salmonella pararyphi j
Escherichia coli communis]
Acrobacicr aerogenes -j
Proeteus vulgaris i
Pseudomonas pvocyauea
Pseudomonas fluor«oens '
1
Stapiij IULOCCUS aureus
Duncan .
Staphj iixxxxus aureus i
pvegciK'; ',
Staphv lococcus albus !
Shjeclla dyt,enlerize (
FSeNner
Shigella dvbenienae Sonne
Salmonella typhi
Salmonella paratyphi ,
Escherichia cohcommunis
•\erob2cter aerogenes
Proteus vjlgaris
Pseudomonas pyocyanea '
Pscu Jornona* fluorescent '
4-CWor-
2.6-di-
brom-
phenyl-
aio-raalo-
nhril
F2223
M 50,000
M 50.000
M 50,000
M 2,503
.M 2.500
M 5.000
M 2.500
M 1,003
M 1,000
M 2.SOO
M 1.000
M' 1,000
4-Chlor-
--nr.rii-
pnenyi-
a/o-rr,aio-
r.i:ril
F 2234
M 50.000
M 50 000
M 50.000
M 5.000
M : -txi
M 25.000
M 5.000
M 5.000
M 2.500
M 2, 1-
azo-mak'-
m:ri)-4-
Karbon-
!n>urc-
aethji-
rster
F'Z21S
M 25.000
NJ 25.000
M 25.000
M 1 000
\J 1 .000
M 5.000
M 2.500
M 2.500
M 2.500
M 2.500
—
, 2-Nhro-
phenyl-
, azo-malo-
1 nitril
. F/2225
M/10,000
: M/io,ooo
M/10,000
M/ 5,000
Mr 5,000
M 25,000
. M/10,000
M, 5,000
: M/ 5,000
' M/ 5,000
. —
—
1-Naphtvl-
t'zo-iiwlo-
Tiiinl
F;'2241
Kl 50.000
M 50.000
M 50,000
«>S 5 WO
M 2.500
M 10.000
M 5.000
M 2.500
M 2.500
M 2,500
M 1 .000
M l.txiO
3-Nilro-
plienyl-
azo-malO"
nitril
F/2226
M/10,000
M/10,000
M/JO.OOO
M' 5,000
M/ 2,500
M/10,000
; M/ 2,500
M/ 2,500
M/ 2,500
M/ 2,500
1 ™"~
I
4-Rtom-
-i-naph-
lyt-azo-
malo-
nilnl
F/2244
M ,'50 ,000
M ,'50.000
M/50.000
M' 5,000
M / 2 500
M,l 0.000
M 5 000
M 2,500
M/ 2,500
M; 5,ooo
M' 1,000
M/ 1.000
4-Nitro-
phenyl-
azo-malo-
nitril
F.2227
M/ 10.000
M 110,000
M.iO.OOO
M/ 5.000
M: 2.500
M 10.000
M- 2.500
' M' 1.000
: M' 1.000
M' 1,000
—
• —
Azoben-
zol-4-azo-
ntalo-
nitri!
F/2245
M/50,000
M/50.000
M/50,000
— ™.
_*_
__
__
: :
„
—
2-NtethyI- 3-Methyl-
4-nitro- ' 4-nuro-
phen>l- ' phenyl-
azo-mato- ! azo-malo-
nitril ( nttri!
F.2228 , F/2229
1
M, 10.000 M/10,000
M/10,000 '• M/10,000
M/J 0,000 i M/10,000
— Ml 1,000
— • M/ 1,000
M' 5.000 ' M' 5.000
M/ 1,000 . M/ 1,000
— : —
1
i . -~
i
! — __
— : —
S.r-Di-
methyl-
dipht'ryl-
en-4.4"-
bis-(azo-
malo-
niiril
F/2247
Mi 10,000
M/10,000
M' 10,000
_—
_—
~__
__
—
Phenyl-
azo-cyan-
CSSjg-
saure
meihvl-
esfer
F/2248
—
_~.
_
™_
___
~
:
„
)
—
_
4-Meih\l-
! 2-nitro-
| phen>i-
i azo-raalo-
! nitni
F 2330
1
; M 10,000
j M 10.000
M 10.000
i
1 M' 2.500
i M 2.500
M 5.000
M 2.500
. M 2.500
: M 2,500
M 1.000
—
—
4-Tolyl-
azol-
azo-rnalo-
nitnl
F.2231
M1 10,000
M '10,000
M. 10,000
M; 1.000
M' liOOO
M 2,500
M 1.000
M 1,000
M' 1,000
M1 1.000
—
—
4-Chlor-
phcnyl-
a^o-cyan-
ess:g-
saurc-
tnethyl-
ester
F/2250
—
.
__
—
2-Chlor-
4-rtiiro-
• phen> I-
azo-malo-
nj'tri!
, F 2232
1
M 10.000
M- 10.000
M 10,000
M; 2.500
—
; M/ 2,500
M' 1.000
—
—
—
—
j —
Lphcnjl-
azo-c>an-
j »sig-
i saurc-
i aethyl
i ester •
!
! F225I
i
__
; — -
:
_
—
__
:
,
™_™,
—
J-Chlor-
4-n:tro-
phcnvl-
azo-malo-
rutnl
F 2233
M 50.000
M 50,000
' M 25.000
M 5.000
, M 2.5CO
M 10.000
\1 2.5CO
• SI 2.500
I M 2.5CO
j M 2.500
• M 1 .000
: M I.OO3
4-lol>l-
^z^}-c> an-
e«:g-
saure-
ae:h>l-
ester
F, 2252
—
.
__^
__
-------
Table 1l|0. Continued
Siaphylococcus aureus Duncan
Staphylococcus aureus pyogenes
Staphylococcus albus
Shigella dysemeriae Flexner
Shigella dysenteriae Sonne
Salmonella typhi
Salmonella paratyphi
Escherichia coli communis
Acrobacter aerogenes
Proieus vulgaris
Pseudomonas pyocyanea
Pseudomonas fluoresccns
> 4-Chlor-
' phenyl-
azo-cyan-
essig-
saure-
aethyl-
esier
' F.2253
—
—
j —
—
—
—
—
' —
—
f —
! —
i —
Phenyl-
azo-cyan-
essig-
saure-
artilid
F/2257
—
—
—
—
—
—
—
—
—
—
—
4-Chlor- Phenyl-
phenyl- I azo-cyan-
azo-cyan- j essig-
essig- I saure-
sa'ure-
anilid
F/2259
(4'-chIor-
anilid)
F/2260
_ | __
— i —
—
—
—
—
—
—
—
'• —
—
—
— —
— I —
— i —
i
4-Chlor-
phenyl-
azo-cyan-
essig-
saure-
(4'-chlor-
anilid)
F/2262
—
—
—
—
—
—
—
—
—
—
—
—
Phenyl-
azo-cyan-
essig-
sa'ure-
hydrazid
F/2263
—
—
—
—
—
—
—
—
—
—
—
—
I 4-Tolyl-
' azo-cyan-
i essig-
4-Chlor-
phenyl-
azo-cyan-
i sSure- essig-
| hydrazid
i
j
j F/2264
i
—
—
—
i —
! —
i
j —
—
—
—
i —
! —
saure-
hydrazid
F/2265 ,
i
M/ 5,000 '
M/ 5,000
M/ 5,000
' —
j
~~ ~ l
— '
'
— '
t
Phenyl-
azo-
acetvl-
aceton
F2266
—
—
— •
—
—
—
—
—
—
—
—
—
3-Tolyl-
azo-
acetyl-
accton
F'2267
—
—
—
—
—
—
—
—
—
—
—
—
Phen> I-
azo-acet-
es^ig-e^ter
F/2271
3-Tolyi-
1 azo-acet-
'• essig-ester
!
i
• F/2272
! 4-Tol> 1-
i azo-acet-
1 essig-ester
,
•
! F/2273
i 3-Chlor-
| phen>l-
1 azo-acet-
; essig-ester
j
: F/2274
4-Chlor-
piienyl-
azo-acei-
essig-ester
F/2275
; Pnenvl-
azo-m^lon- i
i saurc !
! diaethjl- !
: ester |
j F/2776 !
4-Tol>l-
azo-malon-
saure-
diaeihj.1-
ester
F/I277
4-Chlor-
phenvl-azo-
maJonsaure
didcthyj-
ester
F2278
Staphylococcus aureus Duncan
Siapli> loc-^ccus aureus pvogenes
Siaph>iococcus albus
Shjgclia dysenteriae Flexner
Shigella dysenieriae Sonne
Salmonella typhi
Salmonella paratxphi
'ibcherichia coli ccmmunii
j Aerobacter aerogenes
I'roteus vulgaris
Pieudomonas pyocyaaea
iPseudomonas
-------
Table 1M . Azo Compounds a" Tube re ulos tats
Reprinted with permis-
sion from Biochem.
Pnarmacgl. 13:285-318
(1964). Copyright by
Pergamon Press Inc.
1, An
F/2201
1-72202
F/2203
FC204
F/2205
F'2206
F, 2207
F/220K
F/220V
F/2210
F/221 I
F/22I2
F/221 3
F/2214
(722 IS
F/22I6
F/2217
F/2218
IV22I9
I-/2220
F.2221
F/2222
F/2223
J-''P2224
F/2225
F/2226
F/2227
F_/2228
F/2229
F/2230
F/223 1
F/2232
F/2233 '
F/2234
F/2235
F/223G
I '72237
I'/2218
Phcnyl-iv/o-mnlonitril
2-Toh l-a/w-malonitril
3-ToIyl-azo-maIonitril
4-Tti|yl-;u0-malonitril
2-Chfor-plwm I-azo-makwiilril
3-Chlor-pheny!-a70-rrmI0nilriI
4-Clikir-phi'nyl-azo-malonilrH
4-Hrom-plicnyl-azo-m,ilonilriI
4-JtHt-fthen>'!-;uo-malomlril
4-/Veiho\y-phenyl-a70-maloniiril
2-Mcihyl-4-brom-p]icnyl-az0-malomtril
2-Mctliyl-4-jod-pln:nyl-a70-matonMrtl
3-Melliyl-4-brt>ni-plicnyl-azo-malonilnl
4-Meihyl-2-brom-phenyl-a7.o-malonilril
2-Broni-4-aethi.viy-plicnyl-aziMn.ilonitriI
2,5-Dichfor-phcnyl-nzo-rnalonilriI
3,5-Dibrom-phcnyl-a7!vmatonHril
2-Cl>ior-4-b!om-pheayl-.uo-mak>«ilril
j-C'hliir-l-bioin-plicnyl-a/o-maltxiiiril
2-MclIiyl-4,6-dibruiii-p)icnyl-,iA»-(ii,iIi)niuiI
2-C'liloi-4,6-tlibroni-phciiyl'-.i/«-ni.iloniiril
F '22-10
I ','224 1
1 7224 2
I-'.'2MX
IV2244
l'/2245
1-72246
I:/2247
F/224S
F/2249
F/22SO
F/225 1
F/2252
F/2253
4-CIu>r-2,6-vlibron\-i')hcnyl-a/o-inaUmitril
2l41(«-Tnbrojii-phcnyl-a7o-malrt)iitril
2-NUro-p)ieiiyl-a7.o-m.iIonilril
3"Nitro-i)henyl-azo-nulonitri!
4-Nitro-phenyl-azo-iiulonttril
2-Mcthyl-4-nitro-phcnyl-azo-n\alonitril
3-Mcthyl-4-nitro-plii:nyJ-azo-nwlonitriI
4-Mcihyl-2-nitro-phcnyl-azo-malonitril
2-N!iro-4-actho\y-ph€ny!-azo-niak"initril
2-Clilor-4-nuro-phL'nyl-azo-malonitril
3-Chlor-4-nitn>-phcnyl-azo-nialonitril
4-Chlor-2-nilro-phcnyl-azo-niaionitril
4-ALiMy):unino-plicnyI-;i/.O"nMU>niUil
l'hcnyl-a/x)-maloniinl-2-karbonsaiirc
I'ltciiyl-a/o-iii.iloniiiil-'I-karbonsaure
l*licii5'l-;!/i»-i>i.ilonitnl-4-kail;(»iis;iiirc-aclIiyl-csler
3-lly(1ntxy-plicnyl-a/'o-maloni!nl«1-kaibiin<>;iurc
'
pyi iii)idyl)-stiirt)iiaink)
-N,iplilyl-a/(i-nialt>iiilril
IIiciu-l'ii.tplilyl-a/D-iiialt'iiili'il
DiplK-ii>4cii-4.4'-bisj,iA)-nial
-------
Table 1U2. Azo Compounds as Bacteriostats
N5
R
l'
' Bacterium
Staphylococcus aureus
Duncan
Staphylococcus aureus
pyogcncs
Staphylococcus albus
Sh'gella dyscntcriae Flexner
Shinclla dyscnteriac Sonne
Salmonella, typhi
Salmonella, paralyphi
Eschcrichia coli communis
Acrobaclcr acrogencs
Prolcus vulgaris
Ps>cudoinonas pyocyanca
Pscudomonas lluorescens
Staphylococcus aureus
Duncan
Staphylococcus aureus
pyogcnes
Staphvlococcus albus
Shigeila dysenteriae Flexner
Shigelki dysenteriae Sonne
Salmonella typhi
Salmonella paratyphi
H-.chcr it'iia colicommunis
Aerobacter acrogcnes
Proteus vulgaris
Pscudomonas pyocyanea
PbeiKlumomis lluorescens
4-Acetyl-
phenyl-azo-
malonitril
F/2313 ..
M/ 10,000
Ml 10,000
Ml 5,000 ,
Ml 5,000
Ml 5,000
M/10,000
M/ 5,000
Ml 1,000
Ml 1,000
Ml 1,000
—
—
4-(4'-Acetyl-
phenyl-azo)-
3-methyI-
pyrazolon(S)-
yl-I-amidin-
ammo-
guanidon
dihydrochlorid
F/2331
M/ 5,000
M/ 5,000
M/ 2,500
_
—
—
—
—
—
—
—
—
4-Acetyl-
phenyl-azo-
malonitril-
semicarbazon
F/2318
Ml 2,500
Ml 2,500
M/ 2.500
M/ 2,500
Ml 1,000 '
M/ 2,500
M/ 1,000
M/ 1,000
—
—
—
—
4-Phenyl-azo-
3,5-dimethyl-
pyrazolyl-1-
amidm
hydrochlorid
F/2344
M/ 5,000
M/ 2,500
M/ 5,000
Ml 2,500
• M/ 1,000
M/ 1,000
M/ 1,000
M/ 1,000
—
—
—
—
4-Acetyl-
phenyl-azo-
malonitril-
oxym
F/2321
M/ 1000
M/ 1,000
(M/1000)
M/ 1,000
—
—
—
—
—
—
—
•~
4-(3'-Tolyl-
azo)-3,5-
dimethyl-
pyrazolyl-1-
amidin
hydrochlorid
F/2345
M/ 5,000
M/ 5,000
M/ 5.000
M/ 21500
M/ 1,000
Ml 1,000
M/ 1,000
M/ 1,000
—
- —
—
—
4-Acetyl-
phenyl-azo-
malonitril-
phenyl-
hydrazon
F/2322
M/10,000
M/10,000
M/ 5,000
M/ 5,000
M/ 2,500
M/ 5,000
M/ 1,000
M/ 1,000
M/ 1,000
M/ 1,000
—
—
4-(4'-Tolyl-
azo)-3,5-
dimcthyl-
pyrazolyl-1-
amidin
hydrochlorid
F/2346
M/10,000
M/10,000
M/10.000
Ml 2,500
M/ 1,000
M/ 1,000
M/ 1,000
M/ 1,000
—
—
—
— .
4-Acetyl-
phenyl-azo-
malonitril-
salizyloyl-
hydrazon \
F/2323
M/ 5,000
M/ 5,000
M/ 5,000
M/ 2,500
M/ 2,500
M/ 2,500
—
—
—
—
—
— •
4-{3'-Chlor-
phenyl-azo)-
3,5-dimcthyl-
pyrazolyl-f-
amidm
hydrochlorid
F/2347
M/10,000
M/10000
M/10000
M/ 2^500
(M/1000)
(M/1000)
(M/1000)
—
—
— .
—
—
4-Acetyl-
phenyl-azo-
malonitril-
isonikotinoyl-
hydrazon v
F/2324
. . M/ 2,500
M/ 2,500
(M/1000)
M/ 1,000
—
—
—
—
—
—
—
—
4-(4'-Chlor-
phenyl-azo)-
3,5-dimethyl-
pyrazolyl-1-
amidin
hydrochlorid
F/2348
M/10,000
M/10,000
M / 1 o non
M/' 2,500
M/ 1,000
Ml 1,000
M/ 1,000
M/ 1,000
—
—
—
—
4-Acetyl-
phcnyl-azo-
cyanessigsiiure-
melhyl-cster-
amino-
gnanidon
hydrochlorid
F/2328
Ml 2,500
Ml 2,500
M/ 1,000
Ml 1,000
—
—
—
—
—
—
—
—
4-(4'-Tolyl-
azo)-3,5-
dimethyl-
pyrazolyl-1-
thiocarbor-
sunrcamid
F/2356
Ml 2,500
M/ 2,500
M' 2,500
.
—
. .
—
___
—
—
—
4-(4'-Acetyl-
phenyl-azo)-
3,5-dimcthyl-^
pyrazolyl-
1-amjdin-
aminoguanidon
dihydrochlorid
F/2330
M/10,000
M/10,000
M/10,000
—
—
—
—
—
—
—
—
~~~
4-(3'-CMor-
phcnyl-azo)-
3,5-dimcthyl-
pyrazolyl-1-
thiocarbon-
saurcamid
F/235-1
.- Ml 2,500
M/ 2,500
M' 2 ^00
. .
.
. —
—
—
—
—
—
—
Tables 142-143 reprinted with permission from Bicchem.
Copyright by Pergamon Press Ltd. ~~
i'harmacol. 14:1325-1362 (ly6
-------
Table 1U2. Continued
Staptiylococcus aurcus Duncan
Stapiu iococcus aurcus pyogenes
StaphyU'Coccus albus
Shigc&i dyscntcnac Flcxner
Shigolki ilysentcnae Sonnc ,
Salmonella typhi
S,!li;u>nc!Ia puralyphi ,
Esclienchia coli communis
Acrcbactcr uerogeticx '
I'roteus vtilgaris
I'lcuiUvnoruis pyocyanca '
P-'Cutlornonas fluorcsccns
4-(4'-Chlor-
phenyi-azo)- 4-Chlor-
3,5-tlimcthyl- bcnzal-
pyrazolyl- malonitril
ihioc.irbon-
suurcamid
F/2358 F/2371
M/ 2,500 —
M/ 2,500 —
M/ 2,500 —
— —
— —
— —
— —
_ —
— , —
__ —
— —
— —
Benzyl-brom- 4-Chlor-
malonliril benzyl-brom-
malonitrit
F/2398 F/2399
— Ml 1,000
— Ml 1,000
— Ml 1,000
— Ml 1,000
— M/ 1,000
— M/ 1,000
— __
— —
__ —
_. —
— —
— ' —
Benzyl- 4-Ch!or- Phenyl-
malonitril (als benzyl- malonitril (als
Ausgangsstoff) malonitril (als AusgangwtoiT)
Ausgangssioff)
>
— — M/ 1,000
— — Ml 1,000
— — M/ 1,000
— — M/ 2,500
— — Ml 2,500
— — M/ 2,500
— — Ml 2,500
— — M/ 2,500
— — MJf 1,000
— — M/ 1,000
— — Ml 1,000
— —Ml 1,000
-------
Table 1U3. Azo Compounds as Tuberculostats
1. Aryl-azo-Derivate von Alkyl- und Aralkyl-malomtrilen
F/2279
F/2280
F/22SI
F/2282
F/22S3
F/2284
F/28S5
F/2286
F/22S7
F/22S8
F/2280
F/2290
F/2291
F/2292
F/2293
F/2294
F'2295
F,221J6
I-/2297
f-'22'jS
I- 221/;
I-/2100
I-/230I
F/2102
F/2303
F/2304
F/2305
F/2306
F/2307
Phenyl-azo-methyl-malonitril
2-ToIyI-azo-mcthyl-malonitril
3-Tolyl-azo-metriyl-malomlril
4-Tolyl-azo-melhyI-malonitril
2-Chlor-pheny!-u/x>-rnclhyl-rnalonitril
3-Chlor-phcnyl-iuo-meiIiyt-malonitril
4-Clilor-phonyI-azo-mcthyl-malonitril
4-Acilio\y-plicny!-azo-mo(hyl-malonitril
Phcnyl-a/o-aclliyl-iiuloiiitril
2-Tnlyl-a/o-aeIhyl-in. ilunilril
3-1 cil>l-azo-aclhyl-ni.ilonitril
4-'loly!-,i/o-aclhyl-nialonilnl
2-riilor-pheiiyI-a/o-ucthyl-malonitri!
3-Chlor-phcnyl-,\/.o-iicthyl-inalonitril
4-Cli]ur-phcnyl-.uo-;n:thyl-inalonitril
4-Acilio\y-phcnyl-az(i-1ielhyl-ma!onilril
Plicnyl-a/o-hcn/yl-m.tliinilril
2-'! ol>l-axo-bcn/yl-inr-ben/yl)-m;'.lonitril
3- 1 oly!-a/o-M'-cli >r-hcii/yl)-malonitril
4-Tolyl-u/o-(4'-Ji )r-hcn/yl)-rnalonilril
2-Chli>r-phcnyl-azo-(4'-chlor-bcn7.yl)-malonitril
3-Clilur-phcnyl-azo-(4'-chlor-benzyl)- malonitril
. 4-Chlor-phcnyl-;«zo-(4'-chlor-benzyl)-tnalonitril
2. Aryl-azo-bciuoyl-acclone
F/2308 Plicnyl-a/o-beiuoyl-aceton
F/2309 3-Toly 1-azo-bcnzoyl-iu.x'ton
F/2310 4-Tolyl-,i/''>-bcnzoyl-acclon
F/2311 3-Chlor-phenyl-;\/.o-beuzoyl-aceton
F/2312 ~7' 4-Clilor-phcnyl-;i7o-bcn7oyl-aceton
• 3. 4-Acetyl-phenyl-;uo-DcrivaIe von ";tktive Methylen-Gruppe" enthalten-
den Verbimlungen.
F/2313 4-Acx'lyl-phenyl-azo-malonitnl
F/2314 4-Acclyl-phcnyl-azo-cyanessigsaure-methyI-estei
F/2315 4-Acctyl-phenyl-azo-cyanacctamid
F/2316 4-AccIyl-p!icnyl-azo-acctylacc(on > /
F/2317 4-Acetyl-phenyl-azi)-acctcssigcstcr
4. Mil Karbonyl-Reagenticn gebildetc Dcrivate von 4-AcetyI-phenyl-azo-
methylen-Gruppc cntliahcndcn Vcrbindungen
F/2318 4-Acctyl-phenyl-a/o-nialonilri!-scniicarbon
F/2319 4-Acctyl-phcnyl-a/o-maUinitnl-thiosemicarbazon
F/2320 4-Acctyl-phcnyl-a/o-maUmitril-aminoguanidon hydrochlorid
• F/2321 4-Acctyl-phcny]-a/i)-nialonitril-oxym
F/2322 4-Acctyl-phonyl-a7.(>-mak)nitril-plicnyl-hydrazon
F/2323 4-Acctyl-phciiyl-iU.o-malonitril-salizyloyl-hydrazon
F/2324 4-Acctyl-phcnyl-azo-inalonitril-isonikotinoyl-hydrazon
F/2325 4-Acctyt-plicnyl-a/.o-malonitril-azin
F/2326 4-Acctyl-phcnyl-azo-cyancssigsaure-mcthyl-ester-semicarbazon
F/2327 4-Acc(yl-phenyl-a/o-cy;\ncssij;saurc-mcthyl-cstcr-lhioscmi-
carba/.on
F/2328 4-Acciyl-phcnyl-azo-cyancssiBsaurc-rnctliyl-cstcr-amino-
guunidon hydrochlorid
F/2329 4-(4'-Acx-tyl-p!ionyl-azo)-3,5dimctliyl-pyrazolyl-l-thiocarbon-
saurctuuid-lhiivscmicarbazon
F/2330 4-(4'-Acctyl-p!icnyl-a/o)-3,5-dimcthyl-pyrazolyl-!-amidin-
unnnoijuaMidiin dihydrochlorid
F/2331 4-(4'-Acciyl-pln:nyl-a/o)-3-iiKlhyl-pyrazolon-(5)-yl-l-amidin-
iiminoguanidon dihydruthlond
Ml 2,500
Ml 2,500
M/ 5,000
M/ 5,000
M/ 2,500
M/ 5,000
M/ 5,000
M/10,000
M/ 5,000
Ml 5,000
M/ 5,000
Ml 5,000
Ml 2,500
M/ 5,000
Ml 5,000
M/10,000
Ml 5,000
Ml 5,000
M/10,000
M/10,000
M/10,000
M/10,000
M/10,000
M/10,000
M/25,000
M/25,000
M/10,000
M/10,000
M/10,000
M/ 5,000
M/ 5,000
Ml 5,000
M/ 5,000
M/10,000
M/25,000
M/ 1,000
M/ 1,000
M/ 2,500
M/25,000
M/25,000
M/10,000
M/ 5,000
M/25,000 "
M/10,000
M/1,250,000
M/10,000
M/25.000
M/ 5,000
M/10,000
M/100,000
M/25,000
M/10,000
208
-------
Table 1U3. Continued
5. Mil Hydrazin gcbildctc Dcrivale von Aryl-azo-mel)iylen-Gruppe
enthallcndcn Vcrbmdungcn
F/2332 4-PlicnyI-azo-3,5-diarnino-pyrazol M/ 2,500
F/2333 4-(4'-Tolyl-azo)-3,5-dumiiw-pyrazol M/ 5,0(K)
F/2J34 4-(4^Chlor-phenyl-a/o-)3,5-diarnmo-pyrazol ( M/10,000
F/2335 4-I1hcnyl-uzo-3,5-dnnethyl-pyrazol M/10,000
F/2336 4-(4'-Ti>ly|.azo)-3,5-i1imcthyl-pyrazol M/10,000
F/2337 4-(4'-Chlor-plienyl-a/o)-3,5-dimcihyl-pyrazol M/25,000
F/2338 4-Phenyl-azo-3-umino-pyt;uolon-(5) M/10,000
F/2 W 4-(4'-To!yl-;uo)-3-ummo-pyi\uolon-(5) M/ IO,(WO
F/2340 4-(4'-Clilor-phcnyl-a/.o)-3-ummo-pyrazolon-(5) M/10,000
F/2341 4-l>licnyl-azo-3-i)ietliy!-pyrazolon-(5) Ml 5,000
F/2342 4-(4'-'l (ilyl-azo)-3-mclhyl-pyrazolon-(5) M/10,000
F/2343 4-(4'-(~hlof-plicnyI-azo)-3-methyl-pyrazolon-(5) Ml 5,000
6. Mit-Aminoguanidin-hydrochlorid g'-'bildeie Kondensationsprodukte
von Aryl-;tzo-acetylacetonen und Aryl-azo-acetessigestern.
F/2344 4-Phenyl-azo-3,5-dinictliyl-pryiizolyl-I-amidin hydrcchlorid M/ 5,000
F/2345 4.(3'-Tolyl-uzo)-3,5-dimcthyH-amidin hydrochlorid M/ 5,000
F/2346 4-(4'-Toly!-uzo)-3,5-dimi;thyl-pyri«oIyl-l-amit!in hydrochlorid M/ 5,000
F/2347 4-(3'-Clili)r-phenyl-azi))-3,5-dtmclhyl-pyrazolyl-l-«midin
hydrotlilorid M/ 5,000
F/2348 4-(4'-f )il(ir-plicnyl-a/o)-3,5-diniethyl-pyra?oly!-J-ami(Jin
hydnichlorid M/ 5,000
F/2349 4-I'hcivyl-.i/o-3-incthyl-pyraj;olon-(5)-y!-l-amidiii hydrochlorid M/ 2,500
F/2350 4-(3'-Tolyli-azo)-3-mcthyl-pyrazolon-(5)-yl-l-amidin hydro-
chlorid M/ 2,500
F/2351 4-(4'-Tolyl-azo)-3-mcthyl-pyrazolon-(5)-yl-J-amidin hydiO-
chlorid M/ 2,500
7/2352 4-(3'-Chlor-phenyl-azo)-3-me(hyI-pyrjuolon-(5)-yl-l-amilyl-;tzo)-3,5-dimcthyI-pyrazolyl-l-thiocarbonsiiureamid M/10,000
F/2356 4-(4'-Tolyl-azo)-3,5-dimethyl-pyrazolyl-l-thiocarbonsiiureamid M /10,000
F/2357 4-(4'-Ch!or-phcnyl-azo)-3,5-dimethyl-pyrazolyl-l-thiocarbon-
siinrcamid M/10,000
F/2558 4-(4'-C'h!or-phenyl-azo)-3,5-dimethyl-pyrazolyl-o-thiocarbon-
saurctimid M/10,000
8. Verschiedenc nndcrc Azo-Verbindungen
F/2359 4-Tolyl-azo-nitromethan (=Nitro-formaldehyd-4-tolyl-
hyd"ra?on) M/10,000
F/2360 4-Chlor-phcnyl-azo-nitromethan (= Nitro-formaldehyd-4-
chlor-phcttyl-hydrazon) M/ 5,000
F/2361 Phcnyl-azo-dicyandiamin M/ 2,500
F/2362 2-Tolyl-azo-dicyandiamin \ M/ 2,500
F/2363 3-Tolyl-azo-dicyandiamin M/ 2,500
F/2364 4-Tolyl-azo-clicyandiamin M/ 2,500
F/2365 2-Chlo'r-phcnyl-azo-dicyandiamin M/ 1,000
F/2366 3-Chlor-plicnyl-a/o-dicyandianiin M/ 2,500
F/2367 4-Chlor-phenyl-azo-dicyandiamin M/ 2,300
F/2368 2-/Vcthoxy-phcnyl-azo-dicyendiamin M/ 1,000
F/2369 4-Ac!hoxy-phcnyl-a7.o-Oicyandiamin M/10,000
209
-------
Malyuga et al (1971) reported on the antitubercular activity of
some phenylazo-5-salicylic acid and 2-carboxy-4-phenylazonaphthol-l
derivatives, the substituents being on the "phenyl" ring. In order of
decreasing effectiveness in the salicylic acid series were: 4-chloro,
4-bromo = 4-iodo = 4-methoxy = 4-carbethoxy, 2-, 3-, or 4-methyl,
3-nitro = no substituent, 4-nitro, 2-nitro. This order in the naphthol
series was: 4-methyl, 4-methoxy, 2-nitro = 4-carbethcxy = 4-iodo = 4-
chloro, 3-methyl, no substituent, 4-bromo, 4-nitro = 3-nitro.
XI. CURRENT REGULATIONS
There is only one non-dye azo compound for which a use regulation
has been set, azodicarbonamide, 45 ppm in flour. Section III dealt
i
with world-wide practices regarding food, drug, and cosmetic use of azo
dyes.
XII. STANDARDS
No information was found.
210
-------
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230
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APPENDIX
A list of synonyms and tradenames follows for those azo compounds
most frequently seen in the literature—for whatever reason. In the
case of dyes the "entry" name in the alphabetization was occasionally
somewhat arbitary. The source of azobisisobutyronitrile and azodiearbo-
namide was Modern Plastics Encyclopedia 1972-73, Vol. 49: No. IDA, p.
293 (1972). Two sources were used for the remaining entries: Colour
Index, 2nd edition, and Desktop Analysis Tool for the Common Data
Base (1968).
Amaranth
3t 1 038? Ol * * "•» ®
1302 Red CI
1508 Red CI
C.I. 16185 CI
Acid Amaranth CI
Acid Amaranth I CI
Acid Amaranth H CI
Acid Leather Red I2BU CI
Acid Leather Rubine S CI
Alzea Amaranth CI
Amacid Amaranth CI
Amaranth MERCK,CI, LC, NF-»,USP» USP-A
Amaranth A CI
Amaranth 8 CI
Amaranth B.P.C. CI
Amaranth S CI
Amaranth (the dye)
Amaranth BPC CI
Amaranthe CI
Amaranth Extra CI
Amaranth Cake CI
Amaranth S Specially Pure CI
Amaranth USP CI
Amaranth MD CI
Azo Red R CI
Azo Rubine S
S-Azo Rubine
Azo Rubine S.FQ CI
Azo Rubine SF CI
Azo Ruby S
Bordeaux S Extra Cone, A.Export CI
Bordeaux S Extra Pure A CI
C«"aeert Amaranth CI
Certicol Amaranth S CI
CI Icf a Rub!n
-------
Amaranth cont.
£dtcol Supra Amaranth A CI
Eyrocert Amaranth CI
FD and C Red No. 2 MERCK,CI.LC
FD and C Red No, 2-Aluminum Lake CI
Food Bed Z CI
Fruit Bed A Geigy CI
HD Amaranth B CI
HD Amaranth Supra CI
Hexacert Red No. 2 CI
Hexacol Amaranth B Extra CI
Hidacid Amaranth CI
Hlspacid Red AM CI
2—Hydroxy-1,1 '-azonspht hal ene—3, 6,4"— t rlsul fon Ic acid trlsodt'jn salt
Java Amaranth CI
Ktfyaku Amaranth CI
Kayaku Food Colour Red No. 2 CI
KCA Foodcol Amaranth A CI
K| ton Rublne S CI
Llsaamine Amaranth AC CI
Maple Amaranth CI
2, 7-N aphtha lened Isulfonic acid, 3-hydroxy-4-[ (4-sul fo-1-napht hy 1 )azo)
-, tr t sodi urn salt
Naphthol Red B CI
Naphthol Red S CI
Maphthol Red S Cone, Specially Pure CI
Naphthol Red LZS CI
Naphthol Red SI CI
Maphthol Red S Specially Pure- CI
Neklactd Red A CI
Bakuto Amaranth CI
Raspberry Red for Jellies CI
San—el Amaranth CI
Shiklso Amaranth CI
Solar Red 0 CI
l-(4-Sul fo-1-naph thyl aeo )-2-naphthol— 3,6-dl sul fonlc acid trlrodlum
salt CARF
Takaoks Amaranth CI
Tertracid Red A CI
Toyo Amaranth CI
Trlsodlum salt of l-( 4-sul fo-1-napht hyl azo)-2-naphthol-3,6-dI sulfonlc
acid MERCK
Usaccrt Red No. 2 CI
Victoria Rublne 0 CI
Victoria Rublne 0 for Food CI
Whortleberry Red CI
Wool Bordeaux 6RK CI
Wool Red 40F CI
Amlnoazobenzene
C.I. 11000 CI
Amlnoazobenzene LI
p-A«l noazobenzene MERCK
4—Aminoazobenzene
Aminoaxobenzene (Indicator) CI
p~Am!noazobenzol
4—Aminoazobenzol
p-Amlnodi pheny1Ini de
Aniline, p—(phenylazo)- CI
Aniline Yellow CI
Azobenzene, 4—amino—
4-Benzeneazoani11ne
Brasilazina Oil Yellow G CI
Cellitaiol R CI
Ceres Yellow fi CI
C.I. Solvent Blue ? CI
C.I. Solvent Yel low 1 CI
Fast Spirit Yellow CI
Fat Yellow AAD CI
Indultne R CI
01 I Yellow AAB CI
Oil Yellow AB CI
Oil Y«llow AM CI
Organol Yellow 3A CI
p-( Ph«ny I «»o )ani 1 I n*
p—PHenyIazophftnyI a mln#
SOK>*| Is Y*l low 7.0 f |
fl«
-------
o-Aminoazoto luene
C.I. 11160 CI
C.I. U160B CI
Jtalnoazotoluene CI
*~A*f noazotoluene MERCK
2-*A*lno-5-azotolu«ne
4— MIno-2* ,3-dlmethylazobenzene
o-Af
Bra*tlaztna Oil Yellow R CI
C.I. Solvent Yellow 3 CI
2* , 3-Dlmethy l-4-aminoazobenzene
F«»t Garnet GBC base
F««t Yellow AT CI
r*t Yellow 8 CI
HJd«co 01 1 Yellow CI
QAAT
Oil Yellow CI
Oil Yellow C CI
Oil Yellow I CI
Oil Yellow 2R CI
Oil Yellow 21 CI
01 1 Yellow 2681 CI
OU Yellow AT CI
Orgcnol Yellow 2T CI
Somalia Yellow R CI
9«d«n Yellow RRA CI
Toluazoto lul ctl ne
*-T«lutdIn«, 4-{o-toIifl««o)- CI
4-(o-Toljfl»io)-o-tol ul dine
Tul«b*sc Fast G»rn«t GB CI
Tdl*b«i« F*«t Sarn«t GBC CI
C.L Solvent Yelkm 3
HUE Yellow
Aminoazotoluene
Aminoazotoluenc (indicator) • • •
Brasilazina Oil Yellow R
Fast Oil Yellow
Fast Spirit Yellow
Fal Yellow B
Fas» Yellow AT
OU Yellow
OH Yellow I
Oil Yellow 21
Oil Yellow 2681
OH Yellow AT
Oil Yellow C
Oil Yellow 2R
Oil Yellow T
Oil Yellow T
Orgaaol Yellow 2T
Somalia Yellow R
8wd»n Yellow RRA
Wankol YeH«w NL
233
-------
Azobenzene
>j ft it
Azobenzene MERCK,PI,FCH,GCUCP
Azobenzlde GCUCP,PI
Azobenzol PI,MERCK
Benzene, azodl-
Benzeneazobenzene MERCK
Dlphenyldllmlde PI,GCUCP
Azobisisobutyronitrile
N4C8H12
Ficel AZDN-FF
Nitrocel
Poly-Zole AZDN-FF
Porofor N
Vazo
Azodicarbonamide
Ni^CjHi,
Azobisformamide,1,1 * •
Azocel
Celogen AZ
Ficel AC
Kempore
Porofor ADC
Vinyfor AC
23k
-------
Black PN
*S,C,,HZ,.4N«
1743 Black CI
C.I. 28448 CI
Black PN CI
Blue Black BN CI
Brlllant Black BN
Brilliant Acid Slack SNA Cxport CI
Brilliant Acid Black BN Extra Pure A CI
Brilliant Black
Brilliant Black A CI
Bri IIIant Black BN CI
Brilliant Black N.FQ CI
Brilliant Black NAP
Certicol Black PNU CI
Clleta Black B CI
C.I. Food Black 1, tetrasodl uw salt
r.dlcol Supra Black BN CI
Hexaeol Black PN CI
He Ian Black CI
1,7-Haphthalenedisulfonie acid, 4-ae*tanildo-S-hydroxy-6-[[ 7~«u 1 fo-4-
[ ( p-»ul f ophenyl )azo }-l-*aphthy 1 ]»«o ]-, t«trasodlu« salt CI
T*trasoditt» 2-[4-(p-sul fopheny laxo)-7-»ut fo-1-haphthylazo ]-8-ae«tanid>
o-l-Baphthol-3t5-«ll««lfon«t»
Xylen* Black f
Brilliant Black
C.I. 27260 CI
Brilliant black
C.I. Acid Black 3 CI
C.I. Acid Black 3, tetraiodlun salt CI
N.phthol Black 38 CI
Tertracid Brilliant Black B CI
Brown FK
C.I. Food Brown 1
Hul Yellowish Brown
Edicol Supra Brown OH
Golden Brown KBS
Hexacol Brown FK
Consists essentially of • mixture of the disodium
ult of 4, 4'-(4, 6-diwiaino-ni-phenyknebiBazo)
dibenzenesulfonic acid and the sodium salt of 4*
(4, 6-diammo-m-iolyiazo) benzenesulfonic acid
-------
Butter Yellow
C.I. 11029 CI
Anl 1 1 ne, H, M— dime thy l~p-( phenylazo )-
rtzobenzene, p— dimethyl amino—
B«nzen«?azodlmethy lanl 1 Ine
Brilliant Fast Of I Yellow CI
Brilliant Fast Spirit Yellow CI
Brill iant 01 1 Yellow CI
Butter or methyl yellow MERCK
Butter yellow
Ceraalne Yellow GG CI
C.I. Solvent Yellow 2
DAB
D I set hy I ami noozobenzene CDF
01 methyl ami no azo benzene
NfN— Dime thy 1-4- a ml noazobenzene
p— Di me t hy 1 ami no azobenzene MERCK
4-Dlmefhylsmi no azo benzene
4~( N j N— D i met hylamino)a zobenzene
4— tJimethylamlnoazobcnzol
4-Dl»eth>,'la(ninophenyl azobenzene
N,N— Dimethyl— p—pheny I azoani I Ine
Dimethyl Yellow CI
DMAB
Enlal Yellow ZG CI
Fast Oil Yellow B CI
Fat Yellow CI
Fat Yellow A CI
Fat Yellow R CI
Fat Yellow AD 00 CI
Fat Yellow ES CI
Fat Yellow ES Extra CI
Fat Yellow extra cone CI
Graaal Brilliant Yellou CI
Hethyl yellow
01 1 Yellow CI
Oil Yellow D CI
01 1 Yellow G CI
01 1 Yellow 2G CI
01
01
01
01
01
01
Yellow 20 CI
Yellow 2625 CI
Yellow BB CI
Yellow FN CI
Yollow GG CI
Yellow CR CI
Dt Yellow II CI
01 Yellow PEL CI
Qleal Yellow 2G CI
Organol Yellow ADM CI
Orient Oil Yellow GG CI
Petrol Yellow wT CI
Reslnol Yellow GR CI
SHotras Yellow T2G CI
Somalia Yellow A CI
Stear Yellow JB CI
Sudan Yellow GG CI
Sudan Yellow GOA CI
Toyo Oil Yellow G CI
Uaxol Ine Yel low ADS
Yellow G Soluble In Grease CI
C.I. Solvent Yellow 2
HUE Ye!tow-*Reddwh Yellow
Brilliant Fast Oil Yellow
Brilliant Fast Spirit Yel low ..
Brilliant Oil Yellow
Cerasine Yellow GG
Dimethyl Yellow
-------
Butter Yellow cont.
Famt Oil Yellow B
Fat Yellow
Fat Yellow extra cone. ...
Fat Yellow A
Fat Yellow AD OO
Fat Yellow ES extra
Fat Yellow R
Fat Yellow R (8186)
Grasal Brilliant Yellow...
Oil Yellow
Oil Yellow 20
Oil Yellow 2625
Oil Yellow 7463
Oil Yellow II
Oil Yellow BB
Oil Yellow D
Oil Yellow DN
Oil Yellow FN
Oil Yellow G
Oil Yellow 2G
Oil Yellow GG
Oil Yellow GR
Oil Yellow N
Organol Yellow ADM ...
Petrol Yellow WT
Somalia Yellow A
Stear Yellow JB
Sudan Yellow GG
Sudan Yellow GG
Sudan Yellow GG A
Waxoline Yellow AD ...
Yellow G Soluble in Grease
Chrysoidine Base
Chrysoidine Base CI
Chrysoidine Base A CI
Chrysoidine Base B CI
Chrysoidine G Base CI
Chrysoidine J Base CI
Chrysoidine Y Base CI
Chrysoidine Y Base Men CI
Chrysoidine YD Base CI
C.I. Basle Orange 2
C.I. Basle Orange 2, free base CI
C.I. Solvent Orange 3 CI
C.I. Solvent Orange 34 CI
2 , 4-Di a mlnoazo benzene
F»t Brown GG CI
Grasan Chrysoidine CI
Uaxollnc Orange Y CI
237
-------
Chrysoidine R
N4C12H12"HC1
C.I. Basic Orange 1
HUB Dull Yellowish Orange
ARTIFICIAL LIGHT; brighter
Brasilazina Orange 3R
Calcozine Orange RS
Chrysoidine R ..
Chrysoidine R (Biological stain and
indicator)
ChrysokUn* RN
Chrysoidine 3R ...
Chryaoidine 3RN
Chrysoidine RPI
Chrysoidine RRS
Chrysoidine RS
Chrysoidine RS
Diazocard Chrysoidiae R
Pure Chrysoidine RD
Tertrophene Brown CR
Chrysoidine Y Special
N5°6SC29H19'Na2
C.I. Basic Orange 2
HUB Yellowish Orange-»-Orajige
ARTIFICIAL LIGHT: Brighter
Brasilazuia Orange Y
Calcozine Orange YS
Chrysoidine
Chrysoidine
Chrysoidiae A
Chrysoidine B
Chrysoidine G
Chrysoidine GN
Chryaoidioe GS
Chrysoidine HR
Chrysoidine J
Chry»oidine J
Chry*oidine M, PRL & PRR...
Chryioidioe SL
Chrysoidiae SS
Chrysoidine Y
Chrysoidine Y Base New
Chrysoidine Y Special (Biological stain
and indicator)
Chrysoidine YL
Diazocard Chrysoidine G
Leather Orange HH
Nippon Kajjaku Chrysoidine ...
Pure Chrysoidine YD • -
SuRai Chrysoidine
Tertrophene Brown CG
-------
; Coccine
oSjCzoHj ^ . 3Na
1578 Red CI
C.I. 16255 CI
flcidal Bright Ponceau 3R CI
:Acid Brilliant Scarlet 3R CI
Acid Ponceau 4R CI
Acid Red 18 CI
Acid Scarlet 3R CI
Acid Scarlet 3RZ CI
Acid Scarlet 4R CI
Acllan Scarlet V3R CI
Aizen Brilliant Scarlet 3RH CI
Atul Acid Scarlet
Atul Scarlet F
Brl 11 I ant Ponceau
Bri 1 1 iant Ponceau
Brilliant Ponceau
Bri 1 1 i ant Ponceau
Brilliant Ponceau
Brill iant Ponceau
Bri 1 1 i ant Scar] et
Brill iant Scarlet
Brilliant Scarlet
Bri 1 1 Iant Scarlet
Brill iant Scarlet
Bucocid Brl 1 1 i ant
3R CI
CI
3R CI
3RF CI
4R CI
4RC CI
5R CI
4RC Specially
3R CI
4R CI
3R ( Biological
3R Cone
Pure
stain)
Scarlet 3R CI
Caicocid Brilliant Scarlet 3RN
Certlcol Ponceau
4RS CI
CI
CI
CI
CUefa Ponceau 4R CI
Coccine
Coccin Red CI
Cochineal Red ft CI
Cochineal Red 4R CI
Cochineal Red A Specially Pure CI
Colar.ict Ponceau 4R CI
C.I. Acid Re.d 18 CI,MERCK
C.I. Acid Red 18, trisodium salt
C.I. Food Red 7 CI
Curol Bright Red 4R CI
Ddiihiki Brilliant Scarlet 3R CI
Cdicol Supra Ponceau 4R CI
Eurocert Cochineal Red A CI
Fenazo Scarlet 3R CI
Food Red 6 CI
Food Red 7 CI
HD Ponceau 4R CI
HD Ponceau 4R Supra CI
Hexacol Ponceau 4R CI
Hidacid Fast Scarlet 3R CI
Hl3pacid Brilliant Scarlet 3RF CI
Java Scarlet 3R CI
Kayaku Acid Brilliant Scarlet 3R CI
Kayaku Food Colour Red No. 102 CI
Kl ton Scarlet 4R CI
(Cochineal Red A for Food CI
1,3-Naphthalened 1 sulfonlc icid , 7-hydroxy-8-[ (4-»ul fo-1-napht hy 1 )azo]
-, trisodium salt
Naphthalene Ink Scarlet 4R CI
Naphthalene Scarlet 4R CI
Naphthalene Scarlet 4RS
Neklacld Red 3R CI
Neklacld Red 4R CI
New Coccin CI
New Coccine CI
New Coccine Extra Cone. A Export CI
New Coccine Extra Pure A CI
Ponceau 3R CI
Ponceau 4R CI
Ponceau 4RE
Ponceau 4RF CI
Ponceau 4FT CI
239
-------
Coccine cent.
Ponceau 4R Aluminum Lake
CI
Ponceau 4RE. FQ CI
Pontacyl Scarlet RR CI
Purple Red CI
Rakuto flrilliant Scarlet 3R CI
San-el Brilliant Scarlet 3R CI
Scarlet 4R CI
Scarlet 4Rft CI
Strawberry Red A Gelgy CI
Sugai Brilliant Scarlet 3R CI
Symuton Acid Brilliant Scarlet 3R CI
Taksoka Brilliant Scarlet 3S CI
Trlsodium l-(4~sulfo-1-naphthylazo }-2~naphthoI -6, 8-dlsu 1 fonat e
Victoria Scarlet 3R
Victoria Scarlet Red
Congo Red
H
C.I. 22120 CI
Atlantic Congo Red CI
Atul Conqo Red CI
Af.ocard Red Congo CI
l
-------
Evans Blue
T 1824 MERCK
C.I. 23860 CI
Azovan Blue
4,V-BIs[7-( l-anlno-8-hydroxy-2, 4-di sul fo )naphthyl azo ]-3 ,3' -bi t ol y 1
tetrasodium salt MERCK
C.I. Direct Blue 53 CI
C.I. Direct Blue 53, tetrasodlum salt
Diazol Pure Blue BF CI
Dye evens blue CARF
Evans Blue USP, ADI, MERCK, USP-A
Evans Blue dye
1,3-Naphthalenedlsulfonic acid , 6,6'-[ ( 3,3«-d I methyl -4,4'-blpheny 1 y le =
ne )bls(azo )]bls[ 4-amino-5- hydroxy-, tet rasodl um salt
3 ' ~Methyl-4-dimethylaminoazobenzene
N3C, 5Ht 7
Ani 1 I ne, N ,N-d 1 me t hy 1- p~(m- tol yl azo )-
Ant 1 I ne, N, N-dl met hy 1 -4-( m-tolyl azo)-
3'M-DAB
3'Methyl-DAB
4-(N,N-DI«ethylamt no )-3'-i»eth yla zobenzene
4-Dimethylamlno-3'-methylazobenzene CDF
H,N- Dimethyl— p-( m-tolyl azo )ant 11 ne
HDAB CDF
3«-MDAB
3'-Methyl butter yellow
3'— Me thyl -4— dt net hy 1 aminoazobenzene
Methyl Orange
N3OjSC14H15.Na
C.I. 13025 CI
Benzenesu If onl c acid, p— [ [ p-(dl met hylaml no )phenyl ]azo ]—, monosodlum
salt
C.I. Actd Orange 52, monosodlum salt CI
Enlamethyl Orange CI
Gold orange MERCK
Helianthlne B MERCK
KCA Methyl Orange CI
Methy1 Orange CI
Methyl Orange B CI
Methyl orange sodium salt MERCK,CI
Orange III MERCK
Sodium p-dimethy1 aminoazobenzanesulfonate MERCK
Tropaeolln D MERCK
Methyl Red
N,0,C,,Hts
Benzole acid, o—{ [ p-( dlm«thylaml no )phenyl )azo ]-
C.I. Acid Red 2
p-( Dl methyl ami no ) azo benzene— o-earboxy lie acid
Methyl red CI
-------
Neoprontosil
N4OteS3C,.H,».2N«
Bayer 102
6— Ac* tarn! do-4— hydroxy~3-[ (p—tulfsaoylphenyl )azo}—2,7—naphthal *nedlaul
fonie acid dlsodium salt
Azoaulfamlde IECMTN, ADI,MERCK
QI sod I urn 2~( 4*-sulfamylphenylazo )— 7-acet ami do— 1—hydroxynaphthalene~3 ,
6-d!aulfonate MERCK,IECMTN
Drometll MERCK
LeueoneoprontosiI
2,7-Naphthalenedlsul tonic acid, 6~aceta»l do-4-hydroxy~3-[( p-sulfamoyl =
phenol )azo ]~, d I sodium salt
Neoprontosil ADI,MERCK
Neoprontosil sodium
Prontoall S MERCK
ProntosH Soluble MERCK
Streptocid Rubrlm
Streptozon S
Streptozon II
Niagara Blue 21
Blue 2B CI
C.I. 22610 CI
Airedale Blue 2BO
Ai zen Direct Blue
Ananll Blue 2BX
Atlantic Blue 2B
Atul Direct Blue
Azocard Blue ZB
Azomlne Blue 2 It
Belamlne Blue 3B
Bencidal Blue 2B
Benzanil Blue 2B
Benzo Blue BBA-CF
Benzo Blue BBN-GF
Benzo Blue OS CI
Blue 2R salt
Brasi 1 omlna tt lue 2B
Calcominc glue 2B
ChloranUn* Blue 2B
ChJorazol Blue B
Chlorazol Blue BP
Chrome Leather Blue
C.I. Direct Blue 6
C.I. Direct Blue 6,
Cresottne Blue 2B
Di acotton Blue BB
Diamlne Blue 26
Dlasilne Blue BB
01 gph t ami ne Blu
Dtozlne Blue 2D
Diazol Blue 2R
CI
2BH CI
CI
CI
2B CI
CI
CI
CI
CI
CI
CI
CI
CI
cr
CI
CI
CI
2B CI
CI
tetrasodtun salt
CI
CI
CI
BB
CI
CI
CI
Diphenyl Hue M2B
2B
KF
CI
K
H2B
Diphenj/i Blue
Direct Blue A
Direct
Di rer t
Direct
B lue
DI u«
Blue
CI
CI
CI
CI
2B
GS
Direct Blue
Enlanll Blue 2BN
Fenamln Blue 2B
Flxanol Blue ?.B
Hlspamln Blue ZB
Indigo Blue 2B
CI
CI
CI
CI
CI
CI
CI
K«y«ku Direct Blue BB
HI t*ul DI rect Blue 2BN
CI
CI
2L2
-------
Niagara Blue 2B cont.
Naphtawine Blue 2B CI
2,7-Naphthalenedlsulfonle acid, 3»3'-( 4 ,4"~biphenyt ylenebli
(azo ) Jbts[5-amlno—4—hydroxy—, tetrasodlum salt
Niagara Blue 2B CI
Nippon Blue BB CI
Parainine Blue 2B CI
Phenawln* Blue BB CI
Phano Blue 2B CI
Pontamtne Blue BB CI
Tertredirect Blue 28 CI
Vondacel Blue 28 CI
Niagara Sky Blue 6B
N60,»S«CJ4H2..4Na
C.I. 24410 CI.PI
Airedale Blue FFD CI
Amanl1 Sky B lue 6B CI
Amanll Sky Blue FF CI
Atlantic Resin Fast Blue LLGG CI
Atlantic Sky Blue 6B CI
Atlantic Sky Blue FF CI
Atul Dlrtct Sky Blue FB CI
Azlne Brilliant Blue 6B CI
Azocard Blue 6B CI
Belatnine Sky Blue FF CI
Benzatill Sky Blue FF CI
Benzani 1 Supra Blue 2GN CI
Benzo Brillinnt Blue 6BS CI
Braallamtna Sky Blue SB CI
Brilliant Benzo Blue 6BA-CF CI
Calcodur Blue 6GFL CI
Calcodur Begin Fast Blue 6G CI
Caleonltxe Sky Blue FF CI
Chicago Blue 68 CI
Chicago Sky Blue 6R CI
Chloramlne Sky Blue FF CI
Chloranttnc Fast Blue B5GL CI
Chlorazol Sky Blue FF PI ,CI
Chrome Leather Sky Blue GS CI
C.I. Direct Blue 1 CI
C.I. Direct Blue I, tetrasodium salt
Cresotine Blue SB CI
Blacottor, Sky Blue 6B CI
Diaphtamlne Blue BS CI
IMazlno Sky Hue TF CI
DIazol Pure Blue SB CI
DIphenyl Brilliant Blue FF CI
Direct Blue 6B CI
Direct Blue 6BS CI
Direct Blue FF CI
Direct Blue FFN CI
DI rec t Brigh t Blue
Direct Brilliant Blue FF CI
Direct Brilliant Blue MFF CI
Direct Brilliant Sky Blue 6B CI
Direct Pure Blue 6B CI
Direct Pure Blue FF CI
Direct Sky Blue 6B CI
Direct Sky Blue 6BS CI
Direct Sk» Blue FF CI
Direct Sky Blue Gr*«n Shad* CI
Direct Sky Blue GS CI
En I .nil Brilliant Blue FF CI
Fastusol Brilliant Blue L8GU CI
Fenamtn Sky Blue ^F CI
Flxanol Sky Blue FF CI
HNpamln Sky Blue 6b CI
2k 3
-------
Niagara Sky Blue 6B cont.
Ink Blue 6B CI
Japanol Brilliant Blue 6BKX CI
Kayaku Direct Sky Blue 6B CI
Lumlcrertse Blue 4GL CI
Lumicrease Sky Blue 6GUL CI
Mitsui Direct Brilliant Blue 6B CI
Naphtamine SKy Blue DD CI
Niagara Sky Blue 60 CI
Nyanza Sky Blue 6B CI
Paper Blue 6 B CI
Peramlne Sky Blue FF CI
Phenamine Brilliant Blue 6B CI
Pheno Sky Blue 6BX CI
Pontamine Sky Blue
Pontamine Sky Blue 6BX CI
Pontamine sky blue 6x
Pontamine Sky Blue 6BX Greenish CI
Pure Sky Blue 6B CI
Pyrazol Fast Brilliant Blue VP CI
Shiktso Direct Sky Blue 6B CI
Slrius Supra Blue 4G CI
Sky Blue 6B
Solar Blue 4GL CI
Tertrodirect Blue FF CI
Vegentlne Blue CSU CI
Vondacei Blue FF CI
Orange 1
C.I. 14600 CI
Acid Leather Orange I CI
Acid Orangp I
Ai zen Orange 1 CI
Benzenesu 1 fon i c acid, p-[ ( 4-h yd roxy- 1-n aph t hyl )azo]-, sodium stilt
Certiqual Orange I CI
C.I. Acid Grange ?0
Dye orange No. 1 CARF
En lac id Uranqe I CI
F.xt. D and C Orange No. 3 CI
Hi spacid Orange 1 CI
Java Or an ge I C I
Naphthalene Orange 1 CI
a-Naphthol orange MERCK
Neklacid Orange 1 CI
Orange I CI, MERCK
1333 Orange CI
Orange I Extra Cone. A Export CI
Orange IM CI
A.-F. Orange No. 1 CI
Sodium azo-a-naphthol sul f an I late MERCK
4-p-Sul fopheny lazo-1-na ph tho 1 mono sodium aalt CARF
Tertracid Orange I CI
Tropaeolin DOO no. 1 MERCK
Orange G
N,nTSIC,tH,j.2Na
C.I. 16230 CI
Acldal Fast Orange CI
Acid Fast Orange G CI
Acid Fa-t Orange EGG CI
Acid Leather Orange KG CI
Acid Leather Orange PGU CI
Acid Orange G CI
Acid Orange 2G CI
Acid Orange 10 CI
Act Ian Orange GX CI
Aiiac i d Crystal Orange CI
Apocid Orange 2G CI
-------
Orange G cont.
C.I. Acid Orange 10 j
Acidal Fast Orange
Acid Fast Orange G
Acid Light Orange J
Acid Light Orange SX
Acid Orange G
Acid Orange GG
Acid Orange GG
Acilan Orange GX
Apocid Orange 2G
Brasilan Orange 2G
Calcocid Past Light Orange 2G
Cetil Light Orange GG
Crystal Orange 2G
Eniacid Light Orange G
Erio Fast Orange AS
Fast Acid Orange G
Fast Light Orange G
Fast Light Orange GA-CF
Fenazo Light Orange 2G
Hidacid Fast Orange G
Hupacid Fast Orange 2G
Java Orange 2G
Kiton Fast Orange G
Kiton Fast Orange G
Leather Orange GG
Light Orange G
Light Orange G
Naphthalene Fast Orange 2G ...
NekJacid Fast Light Orange GG
Orange G
Di
Orange G (Biological stain)
Orange G (Indicator)
Orange G, BPC
Orange GG
Orange 2G
Solar Light Orange GX
Tertracid Light Orange G
Wool Orange 2G
Xylene Fast Orange G
C.I. Food Orange 4
Acid Light Orange JA Export .
Acid Orange G
Acid Orange GG Crystals
Dolkwal Orange G
Hexacol Orange G
Hexacol Orange GG Crystals
Light Orange AG Cone.
OrangeG
Orange GG
Orange GG Specially Pure
-------
Orange RN
SCuHtz.Na
C.I. 15970 CI
Ac.dine Orange CM CI
Ac!Ian Orange G CI
Acilan Ponceau 4GBL CI
Amacld Brilliant Orange CI
Brl I 1 1 ant Orange CI
Br1 11lant Orange G CI
Brl11lant Orange GN CI
Brilliant Orange GN Type 8019 CI
C.I. Acid Orange 12 CI
C.I. Acid Orange 12, sodium salt CI
C.I. Food Orange 1 CI
Crocelne Orange
Crocelne Orange Y CI
Crocelne Orange 2G CI
Crocelne Orange EN CI
Croceln Orange CI
Hello Orange CAG CI
Hexacol Orange RN CI
Hlspacld Orange CG CI
Klton Brilliant Orange G CI
Kl ton Ponceau 4G CI
Lutetla Orange 2JR CI
Konollte Orange C CI
2—Naph thalenesul Tort i c acid, 6— hydroxy— 5-( pheny lazo)-, sodium salt
Orange G CI
1008 Orange CI
Orange G Food Grade CI
Orange LZS CI
Orange RN CI
Ponceau 40 CI
Segnale Light Orange GR CI
Slloton Orange GR CI
Tertracld Brilliant Qrsr.gs P4G CI
Orange SS
C.I. 12100 CI
C.I. Solvent Orange 2 CI
Dolkwal Orange SS CI
Ext. D and C Orange No. 4 CI
Fat Orange II CI
Fat Orange RR CI
FD and C Oranpc No. 2 LC
Hexacol Oil Orange SS CI
Lacquer Orange V CI
2-Naphthol, l-(o-tol y 1 azo )-
01 1 Orange OPEL CI
01 1 Orange 0' PEL
01 1 Orange SS CI
Oil Orange TX CI
Oletl Orange SS CI
A.F. Orange No. 2 CI
Orange OT*
Orange 3R Soluble in Grease CI
Orange SS LC
Organol Orange 2R CI
Toluene-2-azonaph thoI-2
1-o-Toly I azo-2-naphthol
2L6
-------
Ponceau 2R
1695 Red CI
C.J, 15150 CI
Acldal t'oneeau 3 CI
Acid Leather Red PER CI
Acid Leather Bed KPH CI
Acid Leather Scarlet IRH
Ac|J Ponceau R CI
Acid Ponceau ,?RL CI
ftcld Ponceau Special CI
Acid Red 26 CI
CI
Acid
Aeld
Acid
Acid
Acid
Scarlet
Scarlet
Scarlet
Scarlet
Scarlet
CI
CI
2B CI
2R CI
2RL CI
2R for Lakes CI
2R for Lakes Bluish
Ahcocld Fast Scarlet R CI
Alzen Ponceau KM CI
Amacid Lake Scarlet 2R CI
Calcocld Scarlet 2R CI
Calcolake Scarlet 2R CI
Certicol Poncea« MXS CI
Colacid Ponceau Special CI
C.I. Acid Red 26 CI
C.I. Acid Red Z'j, disodium salt
C.I. f ood Red 5 CI
Dtsodiupi (2,4-dImethylphenylazo )—Z—hydroxy naphtha lene-3,6-d t aul fona te
Dlsodium salt of l-( 2, 4~xyl yl azo )-2-naphthol-3 ,6-d 1 sul fonlc acid
Edlcol Supra Ponceau R CI
Fcnazo Scarlet 2R CI
Hex a col Ponceau ?, R CI
Hexacol Ponceau MX CI
Htdacid Scarlet 2R CI
Kiton Ponceau R CI
Klton Ponceau 2R CI
Ki ton Scarlet 2RC CI
Lake Scarlet R CI
Lake Scarlet 2RBN CI
2 ,7-Naphthalefiedi sul fonlc acid, 3-hydroxy-4-{ ;
dlsodi um sa11
Naphthalene Lake Scarlet
«4-xy lylazo)-,
Hsphthalene Scarlet R
Naphthazine Scarlet 2R
N«U«e(d Red RR CI
New Ponceau 4R CI
Paper Fed HRR CI
Pigment Ponceau R CI
R
CI
CI
CI
Ponceau
Ponceau
Ponce au
Ponceau
Ponceau
Ponceau
Ponceau
Ponceau
Ponceau
Ponceau
Ponce au
Ponceau
Ponceau
Ponceau
Ponceau
Ponceau
G CI
R CI
2R CI.LC
ZDL CI
2RX CI
R (Biological stain)
2R ( Biological stain)
BN/\ CI
2R Extra A Export CI
MX CI
PXM CI
Red
Red R
RR CI
RR Type 8019 CI
RS
CI
CI
Ponceau XyHdine (Biological stain) CI
2h7
-------
Ponceau 2R cont.
D+C Red No. 5 CI
Scarlet R CI
Scarlet 2R CI
Scarlet 2RB CI
Scarl et 2f1L Bluish CI
Scarlet I7KA CI
Tertracld Ponceau 2R CI
Xylidlne Ponceau CI
Xyl (dine red
wo « um 3a
)-2-naPhthol-3,6-di,ulronic acid dlsod.um salt
Ponceau 3R
NzOTS2Cl9H,..2Na
C.I. 16J55 CI
C.I. Food Red 6 CI
C.I. Food Red 6, disodlum salt CI
Dolkwal Ponceau 3R CI
Ext. D and C Red No. 15
External D and C Red No. 15 CI
FD and C Red No. 1 CI,LC
Maple Ponceau 3R CI
2,7-Naphthalenedlsul fonlc acid, 3-hydroxy-4-[ ( 2, 4, 5-t ri me thy 1 phenyl )»'
zo ]-, diaodium salt CI
Ponceau 3R MURCK,CI,LC
Ponceau 3f)N CI
Ponceau 3R Lake CI
Sodium cumeneezo-p-nnpht hoi dlsulfonatc MERCK
Uaacert Red No. 1 CI
Ponceau 6R
.2Na
C.I. 16250 CI
Acidal Crystals Ponceau CI
Acid Leather Ponceau 6R CI
Acid Ponceau 6R CI
Acid Red 6A CI
Colacid Red 6A CI
C.I. Acid Red 44
Crystal Ponceau CI
Crystal Ponceau M6R CI
Crystal Ponceau 6R CI
Crystal Scarlet 6R CI
Crystal Tertracid Ponceau 6R CI
1, 3-Napht halenedi sut fonic acid, 7-hydroxy-8-( 1-napht hy lazo )-, diaodlum
salt
Ponceare 6R
Ponceau 6R CI
Ponceau Crlstallise Extra A Export CI
Ponceau Crystals 6R CI
Ponceau 6R Crystals CI
-------
Ponceau SX
SzCltH,,.2Na
1306 Red CI
12101 Red CI
C.I. 14700 CI
Certlcol Ponceau SXS CI
C.I. Food Red 1 CI
C.I. Food Red 1, disodiun salt
Dye FD and C Red No. 4 CARF
Edlcol Supra Ponceau SX CI
fn and C Rpd No. 4 CFR,CI
Food Red 4 CI
Hexacol Ponceau SX CI
l-Naphthalenesulfonlc acid, 4-hy droxy-3-[ ( 6-9 ul fo-2,*-xylyl )«ro )-
Ponceau SX
Z-( 6-Sul f 0-3,4-xylylazo )-l-naph thol-4-sul f on tc acid, dlsodlum salt
Usaeert Red No. 4 CI
Prontosil
tSCt ZH, j
B«nz«nesul ton amide, p-[ (2,4-d I amlnophenyl )aco]-
Chrysotdln«, 4«-«ulfa»oyI-
2,4-Diamlnoazobenzene—4'—sulfonamlde
p-[(2,4-DI aminophenyl )azo]benzene9ulfonamId«
ProntosI1
ProntoslI red
Bed streptoclde
StreptocJde MERCK
Sulfaehrysoldlnc INN,INN-A
Sulfifiidochrysoidln*
4-Su! fcnyl-2,4-dl anlnoazobenzen*
SuIphiehryioidI ne
Sunset Yellow
1* N&
C.I. 13010 CI
C.I. 13011 CI
Acid Yellow FWA CI
Dcnzene-ju Ifonic acid, p-[ ( p-a ml nophenyl )azo ]-, sodium salt
C.I. Food Yellow 6 CI
C.I. Food Yellow 6, monosodlum salt CI
Hexacol Yellow RFS CI
New Yellow GMF CI
Sunset Yellow CI
11648 Yet low CI
Yellow RFS CI
2U9
-------
Sunset Yellow
Sj,C,»H,z.2N«
C.I. 1598S CI
Acid Yel low TRA CI
Atul Sunset Yellow FCF CI
Canacert Sunset Yellow FCF CI
Certieol Sunset Yellow CFS CI
Ctlefa Orange S CI
C.I. Food Yellow 3 CI
C.I. Food Yellow 3, disodlum salt CI
Dolhwal Sunset Yellow CI
Dye FDC ye Mow lake 6 CARF
Dire FDC yellow No. 6 CARF
Dye Sunset Yellow CARF
Edicol Supra Yellow FC CI
Cnlacid Sunset Yclloy CI
Eurocert Orange FCF CI
FD and C Yellow 6 CI
FD and C yellow lake No. S CARF
FD and C Yellow No. 6 CI,CFR,tC
Food Yellow 6 CI
HO Sunset Yellow FCF CI
HD Sunset Yellow FCF Supra CI
Hexaeol Sunset Yellow FCF CI J|
Hexacol Sunset Yellow FCF Supra CI ™
KCA Foodcol Sunset Yellow FCF CI
Maple Sunset Yellow FCF CI
2-N»phthalene3ul tan Ic acid, 6 —hydroxy—5**[ { p~sulf epheny 1 )azo]
-, dlaodium salt CI
Orange II rt
Orange PAL CI
Orange RSL cone. Specially Pure CI
Orange Yellow S.FQ CI
Para Orange
1-p-Sulfophenylazo-3-naphthol-6-suIfonlc acid, disodlum salt CFR
Sun Orange A Gelgy CI
Sunset Yellow CI
Sunset Yellow FCF LC,CI
Sun Yellow
Sun Yellow Extra Cone. A Export CI
Sun Yellow Extra Pure A . CI
Sun Yellow FCF
Usaccrt Yellow No. 6 CI
1351 Yellow CI
1899 Yellow CI
A.F. Yellow No.5 CI
Yellow Orange S
Yellow Sun
Y«llow SY for Food CI
Tartrazine
S,CltH12.3Na
C.I. 19140 CI
Acid Leather Yellow T CI
Acid Yellow T CI
Acid Yellow 23 CI
Acllan Yellow GG CI
Airedale Yellow T CI
Alsen Tartrarlne CI
Amactd Yellou T CI
D and C Yellow No. 5 CI
Atul Tartrizlne CI
Bucacld Tartrazine CI
Calcocfd Yellow «CG CI
Calcocld Yellow XX CI
Canacert Tartrazlne CI
3~C»rboxy-5-hjfdroxy-l-p--»u I f opheny l-4-p-su 1 fopheny 1 azopy razole
trlsodlum salt MERCK,CFR
Certleol Tartrazol Yellow S CI
CJlefa Yellow T CI
C.I. Actd Yellow 23 CI
C.I. Acid Yellow 23, trlsodlun salt CI
C.I. Food Yellow 4 CI
Curon Fast Ye)low 5G CI
250
-------
Tartrazine
OolkuaV Tartre.'lne CI
Dyt FD and C Yellow No. S CARF
Edicol Supra Tartrazine N CI
Egg Yellow A CI
Erlo Tartrazlne CI
Eurocert Tartrazine CI
FD and C Y«llow 5 CI
FD and C Y«llo» No. S CI ,MERCK,LC,CFR
Fenazo Yellow T CI
Food yellow S CI
HD Tartrazine CI
HO Tartrazln* Supra CI
Hexacert Yellow No. S CI
Hexacol Tartrazlne CI
Hidaztd Tartrazlne CI
Hlspacid Fast Yellow T CI
Hydrazln* yellow MERCK
Hydroxlne Yellow L CI
Kako Tartrazlne CI
Kayaku Food Colour Yellow No, 4 CI
Kayaku Tartrazlne CI
KCA Foodcol Tartrazlne PF CI
KCA Tartrazine PF CI
Kl tor, Yel low T CI
Lake Yellow CI
Lemon Yellow A CI
Lemon Yellow A Gelgy CI
Maple Tartrazcl Yellow CI
Mitsui Tartrazlne CI
Naphtocard Yellow 0 CI
CI
CI
CI
CI
CI
CI
CI
CI
CI
Nektacid Yellow T
Oxanal Yellow T
San—el Tartrazine
Sugal Tartrailne
Tartar Yellow N
Tartar Yellow S
Tartar Yellow FS
Tartar Yellow PF
Tartran Yellow
Tartraphenine CI
Tartrazine CI,MERCK
Tartraztne B CI
Tartrazine B.P.C. CI
Tartrazlne G CI
Tartrazlne N CI
Tartraztne N CI
Tartrazlne 0 CI
Tartrazine T CI
Tartrazine A Export CI
Tartrazlne Extra Pure A CI
Tartrazine FO CI
Tartrazine Lake CI
Tartrazine Lake Yellow N CI
Tartrazine MCGL CI
Tartrazlne NS CI
Tartrazlne 0 Specially Pure CI
tAE.traz.Jjie; XJ< CI
Tartrazine XX Specially Pure CI
Tartrazine XXX CI
Tartrazine Yellow CI
Tartrazol BPC CI
Tartrazol Yellow CI
Tartrlne Yellow 0 CI
Tri sod I um 3-carboxs/—5—hydroxsj—1—p—iu If o phony 1— 4—p-sul fophenylazopyraz3
ole MERCK
Unttertraeld Yellow TE CI
Usjcert Yellow No. 5 CI
Vondacid Tartrazlne CI
Wool Yellow CI
Xylene Fast Yellow GT CI
1310 Yellow CI
1409 Yellow CI
Yellow Lake 69 CI
A.F. Yellow No.4 CI
Yellow No. S FDC CARF
-------
Trypan Blue
N»Oi«S,C3,H;.8.4Na
Blue 38 CI
C.I. 23850 CI
Amani 1 Sky B 1 ue Ff CI
Bencidal Blue 3H CI
Bcnzamine blue MERCK
Bcnzanll Blue 3BN CI
Benzo blue MERCK
Bcnzo Blue 3BS CI
Blue EHB CI
B r a s I 1 am i n a Blue 3 B CI
Chloramine Blue 3B CI
Chrome Leather Blue 3B CI
C.I. Direct Blue 14 CI
C.I. Direct Blue 14, tetraaodium salt CI
Contio blue MERCK
Cresotlne Blue 3B CI
Diamioebluc MERCK
Dlamine Blue 3B
Dianil blue MERCK
Dlaphtanlne Blue TH CI
Diazinc Blue 3B CI
Dlazol Blue 3B CI
Dlphenyl Blue 3B CI
Direct Blue M3B CI
Direct Blue 3 B CI
Hlapamin Blue 3BX CI
2,7-Naphthalenedi3ul fonlc acid, 3,3'-[(3,3'-dlmethyI-4,4»-blphenylyle =
ne )b i 3( azo ) ]bi s[ 5-am tno—4—hydroxy-, tetrasodlum salt
Napht hs/lamine blue MERCK
Niagara Blue MERCK
Niagara fllue 3B CI
Paramine Blue 3B CI
Pontamtnu Blue 3BX CI
Sodium ditolvldl3azobls-8-amIno-l-naphthol-3,6-disutfonate MERCK
Trypan blue CDF
Trypan Blue MERCK,VBB
Trypan Blue BPC CI
Trypane blue ••
Trypan Red
5SsC,zHJ,.feNa
C.I. 22850 CI
2,7-NapMhalenedlsulfonic acid, 4, 4' -f ( 3-su \ f o-4 ,4 • -b iph^ny 1 y 1 ene )b I s
(azo) ]bis[3-amlno-, pentasodlu* •Jalt
Trypan Ked MERCK,CI
-------
TABLE 4—DYES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED.
IDENTIFIED BY MANUFACTURER/ 1971
[Dyes for which separate statistics are given in table 1 are marked below with an asterisk (*); dyes not so maiktd do
not appear in table 1 because the reported data nrc accepted in confidence and may not be published. Manufacturers'
identification codes shown below are taken from table 3. An x signified that the manufacturer did not consent to
his identification with the designated product]
Dye
ACID DYES
*Acid yellow dyes :
*Acid Yellow 17 --- * ---.
*Acid Yellow 73~ - - --
Acid Yellow 25 - - * - - -
Acid Yellow 29-- - ~- - -
*Acid Yellow 34-- - ------ - -
*Acid Yellow 36-- - - - - * - - -
* Ac id Yel low 38- -- '
*AcidYellow40-- *
*Acid Yellow 42 - -*
Acid Yellow 4^ - -
*Ac id Yellow 54 - *
Acid Yellow 63 • - ---/ _-. . „_..
Acid Yel low 65 - -- --- - -
Acid Yel low 73 ----- -_--_* . _» -
*Acid Yel low 76 - • --* --- -
Acid Ye How 77 - - " ----
Acid Yellow 79 *-•
*Acid Yellow 99 /-.,._._
Acid Yel low 114 - -- " ----
Acid Yellow 121 . * -- - -
*Acid Yel low 124 - • -• ---
Acid Yel low 127 --- *-- - -
Ac id Yellow 128 - -.-- * .___
Acid Yellow 129 • - - - -* - -
Acid Yel low 1 35- ~ - *
*Acid Yellow 1S1 *- -
Acid Yellow 152 -- -_-_--^._--- ,
*Acid Yellow 159 - ---
Acid Yellow 174 --,- .
Acid Yellow 175 -
*Acid orange dyes:
*Acid Orange 24 !_.* .
Acid Orange 45 «..-. ,__* ..._,.
Acid Orjngc SI---- .-_- _. _* . .
Acid Orange ^2 --- - --* - -
*Acid Orange 60 — 1 .__„.
Acid Orange 62 _ „ _.'
Acid Orange 63 - - -
Manufacturers' identification codes
(according to list in table 3)
Arc \ry
ACS ACY ATL BDO CMC DUP HN PDC SDH TRC \ IT
ATI
vpr
VPC, YAW.
TDr»
253
-------
TABLED —DYES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED,
IDENTIFIED BY MANUFACTURER/ 1971—CONTINUED
Dye
Manufacturers' identification codes
(according to list in table 3)
ACID DYL'S--Continued
*A( ' orange dyes—Continued
.. id OraiiRe 72
* o-.d Orange 74
Acid Oiange 7(>
Acid Orange 8S
,"•• ,d Uran,;e 86
'Acid Orange 116-
,'.c id Orange 119
Vid Ur.iHRC \'&
A id Uiaiigu 132
C *~'ier acid orange dyes
•Aci i red dyes:
•Acid Ped 1
•"Acid
*Acid
Acid
•Acid
"Acid
A. id
Acid
Acid
Ac, -1
•Ac .d
Acxii
Acid
ACJ J
Ac_t!
•Acid
Ac-d
* Ac ul
Acii
*Acid
•Acid
Acid
"Acid
Acid
Acid
Acid
*Ac-d
*Acid
•Acid
Acid
Acid
•Acid
Acid
*<\cid
Acid
Acid
Acid
•Acid
Acid
•Acid
Acid
/Vc id
Acid
Acid
Acid
Acid
•Acid
Acid
Acid
Acid
Rod
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
4--.
14 —
17 —
18 —
20--
27 —
32 —
33 —
35 —
37 —
42--
52 —
57--
66--
73—
80--
85--
87--
88 —
89--
97 —
99—
100-
106-
111-
114-
115-
119-
133-
134-
137-
138-
151-
107-
175-
178-
182-
183-
186-
191-
194-
201-
211-
212-
213-
266-
309-
GAF.
ACS. CMC, GAP,
TRC.
ACS.
ACS, ALT, TRC.
ACS, ALT, FAB,
TRC.
OUP.
DUP.
ALT, GAP, HST,
AAP, ACS, ACY,
VPC, YAW.
AAP, ATL, BDO,
ACS, ATL, GAP,
ACS, ATL, TRC.
ACS, ATL, BDO,
ACS, ACY, ATL,
ACS.
GAP.
YAW.
AAP, GAP.
ATL, CMC,, DUP,
GAP.
GAP.
ATL, TRC.
AAP, ATL.
ACS, ACY, ATL,
ATL, GAP, ICI.
ACS, ACY, ALT.
SDH.
ACS, ACY, ATL,
AAP, ATL, BDO,
ATL, GAP.
ATL, CMC, FAB,
VPC.
YAW.
ATL.
AAP, ACS, ALT,
ACS, ATL, GAP.
ALT, ATL.
GAP.
TRC.
ACS, ATL, DUP,
ALT.
ACY, ALT, ATL,
ACS, ATL, DUP,
DUP.
DUP.
ACS, ALT, ATL,
CMC, TRC.
ACY, ATL, CMC,
TRC.
CMC, TRC.
TRC.
DUP.
TRC.
TRC.
DUI1, TRC, VPC.
ACY.
ALT, TRC.
TRC.
TRC.
GAP, .IN, TRC, YAW.
TRC, VPC.
ATL, BDO, DUP, GAP, HN, SDH, TRC,
CMC, GAP, PDC, TFlC, VPC, YAW.
PDC, YAW.
GAP, TRC.
CPC.
GA.F, KN, TRC.
DUP, GAP, PSC, TRC, YAW.
ATL, CMC, DUP, GAF, UN, TRC, VPC. YAW.
DUP, GAF, TRC, SDH, YAW.
GAF, HN.
HN, TRC, YAW.
ATL, DUP, GAF, TRC, VPC.
GAF, HN, TRC.
DUP, UN. TRC, VPC, YAW.
TRC.
BDO, CMC, DUP, GAF, HN.
GAF, VPC.
-------
TABLE 4.--DYES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED-
IDENTIFIED BY MANUFACTURER, ]971--CONT1NUED
Dye
ACID DYES--Continued
*Acid red dye*.,- -Conti nued
Other ,»c id red dy
*Acid \ 10. ;-t ilycs:
*Acid Violet 1
*Ac-d Violet 17
Acid Violet 29
*Acid Violet 43---
*Acid Violet 49---
Acid Violet S6---
Acid Violet 58---
Acid Violet 76--~
Other acid violet
*Acid blue dyes:
Acid Blue 1--
*Acid Blue 9
Acid Blue "'O
Acid Blue ^3
*Acid BIu** 25 -
*Acid Blue* ~>7
Acid Blue 29-----
* \cid Blue <'0~~
Acid Blue 43-----
Acid Blue 48
Acid Blue 74
*AciiJ Blue 78
*Acid Blue 80 ---
Acid lUuc «9
Ac id tUuc c>0
Acid Bluv 9*> ---
Acid Blur 102
*Acid Blutr 1 13
*Acid iiJut* 118
* Acid t»iue 1 20
Acid Blue 129
Acid Blue 14S
*»\cid Hluo 153 and
Acid Blue 161
Acid iMuf 165-
Acid Glue 179
Acid Kluc' -^1
*AciJ 3lue ^'30
Acid Kluc ^"51
•
•
•
•
•
»
•
•
9
*
»
•
158A ". -
•
*
*
Manut'jctm ors ' idfnt i fi cat ion codes
(according to l\si in iJhU- .^}
DUP, TRC, VPC.
DUP.
GAF.
ALT, CMG, DUP, GAF, HN, TRC, VPC.
BDO, CMC, GAF.
ACS, ACY, TRC, YAW.
AAP, ACS, ATI., BDO, CMC, GAF, TRC, VPC.
BOO, CMC, DUP, GAF.
DUP, GAF, SDH.
HSH.
ATL, DUP, Id.
CMC.
ATL, HSU, Id.
ACS, ACY, SDH, TRC.
CMC, GAF.
GAF.
ACS.
TRC.
ACS, GAF.
ACS, ACY, ATL, GAF, SDH.
ACS, GAF, SDH.
-AAP, ACS.
GAF.
ACS.
TRC.
ACS, ATL, BOO, CMC, DUP, GAF, UN, TRC, VPC.
M/T, Vil., H'JO, CMC, GAF.
pro,, v\iv.
A ( • ^> •
ACS, M.I, m , BDO, DUP, GAF, ICI, TRC, VPC.
,M ^, MI . ijiji , CMC, CAP.
V.Y, ,';L.
ACS, \CY, A.CL, CMC, UUP, GAF, HN, TRC.
K I,
use .
ACS, ,M.I, IsIK), CMG, GAF, VPC.
C'U .
Al'.s, tit!'1.
All, UiX), Oljp, CAP, ICI, TRC.
ACS, A a, IPC.
ICI .
c,\r .
ACS.
TRC.
ACS, ATL, YAW.
A.CY, IKC.
TRC.
ACS, CAP'.
ACS, ALT, All., BOO, CMG, DUP, FAh, GAF, US', W.
ACS, ATL, UN.
ACS, ACL, GAF, UN.
nup.
CMG.
ACS, UUP.
Uno, CM:, HN, TRC, VPC.
VI'C.
DUP.
GAF.
we.
VPC.
ACS, DUP, TRC.
'I I'C .
-------
TABLE 4.— Dvt3 r«s MUCH U.S. cp.on
'.in SM.ES WERC P;POHTED,
live
I)H ."--font iii'ied
.Kes- -Cont iti_K,t
j u
Other ,..-j 1 hluc .1 -.
•Acid >;ri.-v;n .Uos.
Acid C-r(i:n 1 ---- —
*Aci J C, ,;cn 3 -------
\c id Hi eon 3 -------
•Ac id I, teen 9 ----- --
A..-H! iVc-i'n 12 ------
•Acid Ci. .vi lu ---- -
Acid i HUM 19 ......
* '\cul (..r; MI 7U ------
Acid Grt-en .?? ------
Men! Green JS ------
Acid r.rccMi ^S ------
Ac\J C i-.-on 41 ------
Acid I'reen ^0 ------
Acid Gu'en 58 ---- -
Ac id Ci een 84 ------
Other acid green d;
~^ciJ brown dyes:
Acid Proun ' -------
Acid
•Aci j
Acid
Brown
Acid Broup.
Acjd Pro'rtn
Acid liroMi
Acid BIO'AII
AciJ i.r'iM'
Acid liv:'WH
Acid Bruwii
14
19
22
>J3-
Acid Brown 1:.J
Acid [iio.Ti 158
Acid br.ii.Ti -13
Otlicr .u id brown dves
•Acid black Jycs:
•Acid BiaiA 1--
Acid Bl,
ACS.
ALT.
ACS,
GAJ;.
AL^,
IRC.
ICI,
•\rv ,
TRC.
ALT, ATI,, CMC,, GA1:, UN, 1IS1', TKC, VT(..
•VCY, OUP.
ACY, GAK. TRC.
ACY, GAP.
GAF.
GAP, TnC.
ATL, R!)0, GAF, PDC, Tf;.
ALT, ATL, CMC, CAP, HSU, ICI, TRC, VPC.
VTC.
r,\r.
ALT, VPc.
KM- .
CAP.
\\P,
I'RC.
111)!'.
TIIC .
GAP.
TRC.
ACI,
ACY.
ACY,
D\P.
OAF.
GA! .
ACY ,
AAP,
ACS,
ACS,
\n.,
C,\F.
ACY,
ACS,
CMC,,
!'.UO ,
ACY.
ACS,
CAF.
VPC.
CMH.
ALT,
ACY, DlIP, GAT, TRC, YAW.
CMC.
rw, YAK.
\LT, DUP, CAI , VTC.
«>CS, ACY, ATL, DUP, GAF, UN, I'm: , TKC, YAK.
ACY.
CMC., DUt', GAF.
DUP, 'IllC.
ICI , 'Uc.
VIL, nil!', GAF, UN, I'RC.
lilt!', TRC.
IRC.
ALT, GAP, 'IRC.
ATL. UN, PDC, VPC, YAli.
M I ,
\l I .
-------
TABLED—DYES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED,
IDENTIFIED BV MANUFACTURER, 1971~CONTINUED
Dye
Manufacturers' identification codes
(according to list in table 3)
AZOIC DYES ANU COMeONENTS--Continued
Azoic CornpcBitione--Cantiirjed
Azoic oranxe dyes:
•Azoic Ov..nRe 3
Azoic Orange 10
Other azoic orange dyes-
Azoic reel dyes:
*Azoic Red 1
*Azoic Red 2
Azoic Red 6
Azoic Red 16
Azoic Red 73
Azoic Red 74-- ---
Other azoic red dyes
Azoic violet dyes:
Azoic Violet 1
Other azoic violet dyes-
Azoic blue dyes:
Azoic Blue 2
•Azoic Blue 3
Azoic Blue 6
Azoic Blue 7
Azoic Blue 8
Other azoic blue dyes—
Azoic green dyes:
Azoic Green 1
Other azoic green dyes--
Azoic brown dyes: 3
Azoic Brown 3
Azoic Brown 7
•Azoic Brown 9
Azoic Brown 10
Azoic Brown 26---
Other azoic brown dyes--
*Azoic black dyes:
Azoic Black 1
Azoic Black 4
Azoic Black 15
Other azoic black dyes—
Azoic Diazo Components, Basea
(Fast Color Bases)
Azoic Diazo Component 2, base--
Azoic Diaio Component 3, base--
•Azoic Diazo Component 4, base--
Azoic Dinzo Component 5, base--
Azoic Diazo Component 8, base--
Azoic Dtazo Component 10, base-
Azoic Diazo Component 12, base-
Azoic Diazo Component 13, base-
Azoic Diazo Component 14, base-
Azoic Diazo Component 20, base-
Azoic Oiazo Component 28, base-
*Azoic Diazo Component 32, base-
Azoic Dnzo Component 34, base-
Azoic Diazo Component 44, base-
Azoic Diazo Component 46, base-
Azoic Diazo Component 48, base-
•\LL.
BUC.
ATL.
ALL,
ALL,
ATL,
ATL.
CAP.
GAP.
ALL,
ATL,
ALL.
ATL.
ALL,
ATL.
CAP.
ALL.
ALL,
ATL.
ALL,
x.
ATL,
ALL,
BUC.
CAP.
ALL,
HST.
ATL,
CAP.
ALL,
ATL,
BUC.
ALL,
GAP,
SDH.
BUC,
BUC,
BUC.
AAP.
ALL,
ALL,
AAP,
SDH.
BUC.
ATL.
CWN,
ATL, BUC, X.
ATL, BUC, x.
ATL, BUC, CAP, X.
BUC, x. .
ATL, x.
BUC, GAP.
ATL. BUC, GAP, HST, x.
ATL.
BUC, VPC.
BUC.
ATL, BUC, GAP, riST, VPC, X.
ATL, GAF, VPC.
BUC, GAK.
ATL, GAF, VPC.
BUC.
BUC, GAF, SDH.
SDH.
GAF.
SDH.
GAF.
BUC, GAF.
ALL, ATL, BUC, SDH.
GAF.
-------
TABLE 4.—DYES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED,
IDENTIFIED BY MANUFACTURER/ 1971--CoNTIi,UED
Dye
Manufacturer ' identification codes
(according to list in table 3)
AZOIC DYES AND COMPONENTS--Continued
Azoic Diazo Components, Salts
(Fast Color Salts)
"Azoic
A? i ; c
•A.'oic
*A" OiC
*Azo)C
•AZLIC
*Azoic
*Ai 01 c
A:< c
AZ--IC
A.-,,iic
.-.'..' C
A? . . ' c
Azoic
Ai >^c
A/. • • r
Az, ic
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
Diazo
azoic
Component 1, salt
bait
salt
Component 2,
Component 3,
Component 4, salt -------
Component 5, salt -------
Component 6, salt -------
Component 8, salt -------
Component 9, salt -------
Component 10, salt ------
Conponent 11, salt ------
Component 12, salt ------
Component 13, salt ------
Component 14, salt ------
Component 20, salt ------
Component 28, salt ------
Component 32, salt ------
Component 34, salt ------
Component 35, salt ------
Comoonent 36, salt ------
Component 37, salt ------
Component 41, salt ------
Component 42, salt ------
Component 44, salt ------
Component 48, salt ------
Component 49, salt ------
Component 1M, salt -----
diazo components, salts-
Asoia Coupling Components
(Naphthol AS and Derivatives)
Azoic
Azoic
Azoic
Arcic
*Azoic
Azoic
Azoic
Azoic
Azoic
Azoic
Azoic
Azoic
Azoic
Azoic
•Azoic
AZOJ c
Azoic
Azoic
Azoic-
Azoic
Azoic
*Azoic
Azoic
Azoic
Other
Coupling Component 2
Coupling Component 3
Coupling Component 4
Coupling Component 5
Coupling Component 7
Coupling Component 8
Coupling Component 10
Coupling Component 11
Coupling Component 12
Coupling Component 13
Coupling Component 14
Coup Ling Component 15
Coupling Component 16
Coupling Component 17
Coupling Component 18
Coupling Component 19
Coupling Component 20
Coupling Component 2]
Coupling Component 20
Coupling Component 34
Coupling Component 35
Coupling Component 43
Coupling Component 44
Coupling Component 107
azoic coupling components-
AAP,
ALL,
AAP,
ALL.
AAP,
AAP,
AAP,
AAP,
ALL,
AAP,
AAP,
AAP,
AAP.
ALL,
ALL,
ALL,
ALL,
BUC,
AAP,
GAP.
ALL,
GAP.
ALL,
BUC,
AAP,
GAP.
SDH.
ATL,
BUC.
ATL,
BUC.
ALL,
ATL,
ATL.
ATL,
BUC.
GAP.
ATL,
ALL,
BUC,
ATL,
ALL,
BUC,
ATL,
ATL,
ATL,
ATL,
ALL,
ATL,
PCW.
11ST.
AI'L,
ALL, BUC, GAP, SOU.
BUC.
ALL, BUC, GAP, SDH.
ALL, BUC, GAP. SDH.
BUC, GAK
ALL, BUC, GA,'.
ALL, BUC, GAP, SDH.
BUC, GAP.
ALL, BUC.
ALL, BUC, GAP, SDH.
ALL, BUC, GAP, SDH.
BUC.
BUC, GAP, SDH.
SDH.
GAP.
GAP.
GAP.
BUC.
BUC.
SDH.
ALL, BUC, GAP.
BUC, GAP.
BUC, GAP.
BUC, HST, SCH.
DUC.
BUC.
BUC.
BUC, GAP.
GAP.
BUC.
ATL, BUC, GAP.
GAP.
BUG, GAP.
BUC.
BUC.
BUC.
BUC.
BUC, GAP.
GAP .
-------
TABLE4. --DYES FOR WHICH tJ.S. PRODUCTION OR SALES WERE REPORTED.
IDENTIFIED BY MANUFACTURER/ 1971--CONTINUED
Dye
BASIC DYLS
dBa =ic yellow dyes:
Basic Ye How 37 --• - ,-,---------
itasic orange dyes ;
."/ -•• ran£e
. 77
. ' k
, a.^
Basic red dyesr
Basic Red 17 *
Basic Red 18 * -...._
Basic Red 22
Bisic Red ">3 *--
Basic Red 29 - - --
Basic Red 48
8: sic violet dyes
Basic Violet 13 -- ..
Basic Violet 14 -
Basic Violet 15 -
'Basic Violet 16
Bj^ic Violet 18 -*
Basic Violet 24
Basic Violet 27 —
Manufacturers' identification codes
(according to list in table 3)
DUP.
ACS, ACY.
ACS, ACY, ATL, DUP, FAB, GAP, TRC, VPC.
ACS, ATL, DUP, GAP, VPC.
DUP.
VPC.
BAS.
BAS.
ACY.
VPC.
DUP, VPC.
DUP.
ACY.
ACY.
DUP.
ATI, DUP, EKT, GAP, VPC.
ACS, ACY, DUP, GAP, PSC, TRC.
ACS, ACY, DSC, DUP, GAP, PSC, TRC.
GAF.
ACS, ACY, ALT, ATL, DUP, FAB, GAF, TRC, VPC.
ACS, GAJ-1.
DUP.
DUP.
DUP.
VPC.
VPC.
ACY.
DUP.
ATL.
BAS, DUP.
ACS, DUP.
ACY, DSC, HSC.
DUP.
ACS, ATL, GAF, TRC, VPC.
ACS, ACY, ATI,, DUP, GAF, VPC.
ATL, DUP, GAF, TRC.
itlUP.
DlJP. '
AfL, DUP, GAF, VPC.
DUP.'
ACY, TRC.
VPC .
BAS.
ACY. • ,
DUP. • ' , ' •„
PUP, GAF: •
ATL, DUP, EKT, VPC.
ACS, ACY, DSC, DUP, IISC.
DSC.
,ACS, DSC, DUP, SDH.
DSC, DUP.
,ATL, GAF.
, ACY, DUP, GAF.
DSC.
ACY, DSC! •
DUP.
AIL, DUP, FAB, GAF, TRC, VPC.
ACY. ,
DUP.
A'fL. '
259
-------
TABLE 4.—DYES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED, /
IDENTIFIED BY MANUFACTURER, 1971—CONTINUED
Dye
Manufacturers' idenl ification codes
(according to list in table 3}
BASIC CYES--Continued
•Basic blue dyes:
'Basic Blue 1
Basic Blue 2
B.isic Blue 3-
Haste Hluc 4 ----,-.,
'Basil. BUiL- 5
Basic Clue 6
*B,T,ic Blue 7
basic Blue 9
Basic Blue 11
Basic Blue 21
Basic Bluf 22
Dasic Blue 26
Basic Blue 35
Basic Blue 41
basic Blue 45
Basic Blue 47
Basic Blue 54
Basil. Blue bO
Basic Blue 69
Basic Blue 75
Basic Blue 76
Basic Blue 77
Basic Blue 82
Basic Blue 87
Basic Blue 97
Other basic blue dyes--
tfasic green dyes:
"Basic Green 1
Basic Green 3
•Basic Green 4 * —
Basic Green 7
Other basic green dyes-
Basic brown dyes:
'Basic Brown 1
Basic Brown 2
'Basic Brown 4
B isic black dyes :
Basic Black 9
Other basic black dyes-
--I-T-
DIRLCT DYES
•Direct yellow dyes:
•Direct Yellow 4--
•Direct 'iellow S--
•Direct Yellow 6--
Dircct Yellow 7--
Direct Yellow 8--
Direct Yellow 9--
'Direct Yellow 11-
•Direct Yellow 12-
fHrect bellow 20-
Direct Yellow 23-
iHrect Yellow 26-
Direct YelloK 27-
•lurect Yellow 16-
*Ui rect \ellow 29-
Dircct 'lei low 34-
Direct bellow 39-
Direct \clU
41-
•nireci \e\low \\-
•IHrect \ellow 30-
Direct Iellow 59-
DSC,
DSC.
ACY,
DUP.
DSC,
ACY.
DSC,
ACS,
' DSC,"
DUP.
ACS,
' DSC,
DUP.
TRC.
VPC.
VPC.
ACY,
GAP.
VPC.
EKT.
ACY.
DUP.
" DUP,
DUP.
DUPl
ALT",
ACS,
DUP.
ACS,
DSC.
VPC.
ACS,
GAP.
ACS,
VPC.
ALT,
ACS,
ACS,
ACS,
ATL.
PCS,
ATL.
ACS,
ACS,
TRC.
DUP.
ALT,
OAF.
ACS,
ATL,
ALT,
TRC.
ATL.
ACS,
A I.I ,
DUF.
GAP,
DUP,
SDH,
DUP,
ACY,
nup,
DUP,
UUP,
BAS.
TRC.
BAS,
ACY,
ACY,
ACY,
ACY,
DSC.
ACY,
ACY,
ACY,
ATL,
ACY,
ATL,
ATL,
ATL,
DUP,
UN.
M.I ,
ATI. ,
SDH, VPC.
GAP, HST.
VPC.
SOU.
DUP.
SU11.
VPC .
SDH.
DUP, EKT, VPC.
DSC, DUP. .
DSC, SDH, VPC.
DUP, GAP, PSC, TRC.
DSC, nUP, GAF, PSC, TRC.
ATL, DUP, GAF, UN, TRC, VPC.
GAF.
DUP, GAF, TRC.
GAF.
ALT, DUP, GAF, UN, IRC, VPC.
DUP, FAB, CAF, TRC.
UN.
?
DUP, GAF, PDC, TRC.
GAF.
AIL, DUP, FAB, GAF, IIS, IRC, VPC
1HIP, FAB, C\F, UN, IISH, TRC, VI'l.
260
-------
TABLE 4.—DYES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED,
IDENTIFIED BY MANUFACTURER, 1971—CONTINUED
•Direct ye! low dy
'Jirect i'o' l.u '
Direct Yellow
•Direct Yei :-M
lUr.-ct Yellow
•Direct Yellow :i)D
YV l.iw
iV 1 low
•Direct
Direct
Direct Yellow
Pire'-t Yellow
Direct Yollow
Direct Yellow
Direct Yellow 120
Direct Yellow
Direct Yellow 123
Direct Yellow
Direct Yellow 127
Direct Yellow
Direct Yellow 132
Other direct yell
'Direct orange dyes:
•Direct Orange
Direct Orange 6
•Direct Orange 8
Direct Ora-.ige 10
Direct Orange 11
•Direct Orange
'Direct Orange 26
'Direct Orange 29
'Direct Orange
'Direct Orange 37
'Direct Orange
Direct Orange i>9
Direct Orange 61
Direct Orange 67
'Direct Orange 72
•Direct Orange 73
Direct Orange 74
Direct Orange 78
Direct Orange 79
Direct Orange 80
Direct Ornng
Direct Orange 83
Direct Orange 38
•Direct Orange 102
Direct Orange 110
Other direct o
•Direct red dyes:
•Direct Red 1--
•Dircct Red 2--
•Direct Red A
Direct Red 5
Direct Red 7
•Direct Red 10--
•Dircct Red 13--
Direct Red 16--
Direct Red 20--
•Diiect Red 23--
• Direct Red 24-.
•Direct Red 26--
'Direct Red 2S--
•Llirect Hod 31--
Direct Red 32--
•Direct Red 37--
Dye
PIRLC'I UYl.S--Continued
0..--1 iint inucd
.,, . .. .•
•
l,t^ , ... --__._„_._»,.«._.
ii7 __,._, ._.,.._-_._.-*--._ _ _-..
12*, . _.__._-._.-._».--____»._-.__.-_
197 __--- - -.,-._---.--_-
131 , ._.__-„---._ ___.»_
es :
6 :
8_ '
10 *
11 • .
15 .
7A „ -* -
-° 3 --
29 » -^. .
34. _ ___*_. _
3;...* .'_
•59 '_
1,9 . - * . J
6j_, ____.. ._-..__-__**.__-„_._
57 ..
72
73_ _•_
74 !
78. •
70 ;
ao •
8i_ *
85 *
S8 . •
102 -- -
aui - -
uo , .
•
•
•
•
•
*
•
•
•
•
*
.
•
*
/
•
Manufacturers' identification codes
(according to list in table 3}
DUP
mm IP FAR HW TBr VPT
Ape
APY
TAF
ACS ACY DUP GAP HN TRC
261
-------
TABLE4- —DYES FOR WHICH U.S. PRODUCTION OK SALE? WEr:F. REPORTED,
IDENTIFIED BY NAflUrAC'fUnER, V:? V -CoNYiiiU£D
Dye
DIRECT DYES--Continued
"Direct red dyes--Continued
"Direct Red 39---
Direct Red 46
Direct Red 62
•Direct Red 72-
Direct Red 73
'Direct Red 75
Direct Red 76
•Direct Red 79 -
•Direct Red 80
•Direct Red 81
Red 95--
Rcd 100-
Red 111-
Rcd 117-
•Direct Red 83-
Direct Red 84-
Direct
Direct
Direct
Direct
Direct Red 120 *-.
•Direct Red 122 -*-
•Direct Red 123 -*-
Direct 127 and 127A -*-
Direct Red 139 *-
Direct Red 149 -
Direct Red 152 -*
Direct Red 153 -"-
Direct Red 209 *-
Direct Red 212 -*-
Direct Red 236
Direct Red 238
Other diicct. red dys%
iirect violet clyes:
Direct Violet 1-- -*--•
•Direct Violet 7 *--
g _•__.
•Direct Violet
Direct Violet 14
Direct Violet 22
Direct Violet 47
Direct Violet 48
•Direct Violet 51
Direct Violet 62
Direct Violet 66
Direct Violet 67
Other direct violet dyes
•Direct blue Jyes:
•Direct Blue 1
*r>j££st Blue 2
'Direct Blue (>-
*0i'cct Blue 8
Direct Blue 14
•Direct Blue 15
•Pirect Blue 22
Tired Blue 24---
•Direct Blue 25
Direct Blue 26
•direct Blue 67
•Direct Blue 71-~
Direct Blue 74
Direct Blue 75
•Direct Blue 76
'Direct Blue 78
•Direct Blue 80
Direct Blus 81 --
•Direct Blue 86
Munuf actu i ors ' identification i_oc
(according to Ust in table 3)
ATI., DUP, GAP,
ML.
ATL, TRC.
ACS, DUP, GAP,
ACS, ATL.
ACS, ATL, CMC,
GAP.
ATI., CMC, UN,
ACS, ALT, ATL,
ACS, ACY, ALT,
VI'C, YAK.
ACS, ALT, ATL,
ATL.
VPC.
ATL.
GAP.
DUP.
CMC, VPC.
ATL, CMC, TRC,
ATL, CMC, GAP.
ATL, CMC.
ATL, VPC.
ATL, CMC, DUP.
CMC.
ATL, CMC.
TRC, VPC.
VPC.
DUP.
DUP.
ALT, ATL, GAP,
ACS, ATL.
ACS, ATL, GAP.
ACS, ATL, DUP,
ACS, ATL,
DUP.
GAP.
ACS.
ACS, ATL, DUP.
ACY.
ATL, TRC.
UUP.
ALT.
AAP, ACS, ACY,
AAP, ACS, ATL,
AAP, ACS, ACY,
ACS, ALT, ATL,
ACS, ATL, TRC.
ACS, ATL, DUP,
ACS, ATL, CMC.
ATL, UN, YAW.
ACS, ATL, GAP,
ATL.
ACS, ATL, DUP,
ACS, ML, GAP,
DUP.
TKC.
ACS, ALT, ATL,
ACS, ATL, CMC,
ACIi, ALT, ATL,
ML.
AAP, Af:>, AIT,
IIIC, VPC
TRC, YAW.
TRC,
GAP.
TRC, VPC.
BDO, CMC, FAB, UN, HSU, SDIl, fRC, VK,
ATL, CMC, DUP, GAP, UN, IISH, TRC,
FAB, HN, HSH, TRC, VPC.
VPC.
HN, HSH, TRC.
GAP, UN, TRC.
ATL, [)UP, GAP, HN, TRC, VPC, YAH.
DUP, rtB, GAI;, 11N, HSH, TRC, Vt'Ll, YAW.
ATL, DUP, GAP, HN, HSH, TUC, YAK.
DUP, GAP.
GAP, VPC, YAW.
TRC, YAW.
TRC.
TRC, VPC.
C.AP, UN, HSH, TRC, VPC.
DUP, TRC.
HUP, FAB, GAP, HN, HSH, TKC, VPC.
\I'L, I)1 IP, PAD, rM, UN, ICC, Slid,
-------
TABLE 4.—DYES FOR WHICH U.S PRODUCTION OR SALES WERE REPORTED/
IDENTIFIED BY MANUFACTURER/ 1971—CONTINUED
Dye
Manufacturers' identification codes
(according to list in talile 3)
DIRECT DYES--Continued
'Direct blue dyes--Continued
Direct Blue 87
Direct Blue 91
•Direct Blue 98--
100-
Direct blue
Direct blue 104
•Direct Blue 120, 120A---
•Direct Blue 126---
Direct Blue 136
Direct Blue 143
Direct Blue 151
Direct Blue 160
Direct Blue 189
Direct Blue 191
Direct Blue 199
'Direct Blue 218-
Direct Blue 224
Direct Blue 238
Direct Blue 263-
Other direct blue dyes--
•Direct green dyes:
•Direct Green 1
•Direct Green 6
Direct Green 8
Direct Green 26
Direct Green 27
Direct Green 28
Direct Green 38-?
Direct Green 39
Direct Green 45
Direct Green 46
Direct Green 47
Direct Green 51
Direct Green 69
Other direct green dyes-
•Direct brown dyes:
Direct Brown 1
•Direct Brown 1A
Direct Brown 2
Direct Brown 3
Direct Brown 6
•Direct Brown 31
Direct Brown 32
Direct Brown 33
Direct Brown 40
Direct Brown 44
Direct Brown 48
Direct Brown 59
•Direct Brown 74
•Direct Brown 95- -
Direct Brown 106
•Direct Brown 111
Direct Brown 112
•Direct Brown 154
Direct Brown 218
Other direct brown dyes-
•Direct black dyes:
•Direct Black 4 --
Direct Black 8
•Direct Black 9
Direct Black 17
•Direct Black 19-- -
*l)in-ct Black 22--
-Direct Black 36
ICU
TRC.
ATL,
ALT,
DUP.
ATL,
ATI,,
GAP.
DUP.
ATI,,
TRC.
TRC.
AAP,
DUP,
ACS,
ATL.
ACY.
DUP.
ALT,
AAP,
AAP,
TRC,
DUP,
DUP,
TRC.
DUP,
GAP.
ATL.
VPC.
ATL,
TRC.
TRC.
ACY,
ACY,
GAP,
AAP,
VPC.
TRC,
AAP,
GAP.
DUP.
AAP.
GAP,
AAP.
YAW.
AAP,
ACS,
GAP.
DUP,
ATL.
ACS,
ACS.
ALT,
ACS,
TRC,
ACS,
GAF.
ATL,
ALT,
AAP.
GAF, TRC, VPC.
ATL, UN.
DUP, FAB, HN, TRC.
DUP, HSH, TRC, VPC.
TRC.
ACS, ALT, GAF.
GAF, HN.
ALT, ATL, DUP, FAB, GAF, HN, TRC, VPC.
GAF, VPC.
ACS, ACY, ATL, FAB, GAF, TRC, YAW.
ACS, ATL, FAB, GAF, HN, TRC, YAW.
TRC.
TRC.
GAF.
DUP, GAF.
ALT, DUP.
ATL, HN.
TRC, YAW.
ACS, ACY, ATL, DUP, GAF, HN, HSH, TRC, YAW.
YAW.
ACS, ATL, DUP, GAF, TRC, YAW.
YAW.
ACS, DUP.
ATL, DUP, FAB, GAF, UN, HSH, TRC, YAW.
GAF, TRC. .
DUP, FAB, TRC, YAW.
ATL, HN, HSH. VPC.
ATL, GAF, HN, TRC, YAW.
YAW.
ATL, DUP, HN.
GAF, HN, THC.
ATL, GAP, HN, TRC, VPC, YAW.
263
-------
TABLE*. —DYES FOR WHICH U,S, PRODUCTION OR SALES WERE REPORTED,
IDENTIFIED BY MANUFACTURER/ 1971--CONTINUED
Dye
D1R1.CT DYES- -Continued
•Dijf. - Mack d)'es--('ont inued
D1SPERSF: DYES
*0ispeiic yellow dyes:
*t)iS(H'rse orange dyes:
Manufacturers' identification codes
(according to list in tasle .1)
AAP.
ACS,
TRC,
AAP,
ACS,
ATL,
GAP,
ACS,
AAP,
ACS,
ACY,
GAP,
DUP.
AAP,
GAP,
TRC,
AAP,
GAP,
0UP.
AAP,
AAP,
AAP,
TRC.
AAP,
HST.
HST.
DUP.
HST.
ACY.
VPC.
VPC.
EKT.
AAP,
EKT.
EKT,
EKT.
VPC.
VPC,
VPC,
AAP.
SDC.
EKT,
AAP,
AAP,
HST,
AAP.
AAP,
TRC,
DUP,
AAP.
AAP,
ICC,
ALT,
TRC.
IRC.
Dili1 .
UUP.
i.u.
ACY, K\B, GAP, HN, HSH, TRC, YAK.
ACS, DUP, GAP, TRC.
TRC.
UN.
ACS, ATL, FAB, HN, HSH, TRC, YAW.
HN 1 RC
ALT, ATL, HSH, TRC, YAW.
ICI.
ALT, OUP, GAP, HN, HSH, ICC, TRC.
HN, ICC.
ALT DUP, EKT GAP HN ICC TRC
EKT, GAP, ICC, TRC.
EKT, ICC.
ALT, BUC, DUP, EKT, GAP, HN, ICC, MAY, SDC, TRC,
DUP, GAP, ICC, SDC, TRC.
EKT.
MAY, SDC, TRC, VPC.
DUP, GAP, UN, HSH ICC, TRC,
BUC, LK1, GAP, ICC, SDC.
EKT, GAF, HN, HSH, ICC.
EKT, UN, TRC,
GAF.
TRC.
HST.
26U
-------
TABLE 4.--DYES FOR WHICH U,S, PRODUCTION OR SALES WERE REPORTED/
IDENTIFIED BY MANUFACTURER, 1971—CONTINUED
Dye
Manufacturers' identification co
(according to list in table 3)
DISPERSE DYES—Continued
•Disperse orange dy«s--Contiuued
Disperse Orange 58 AAP, EKT.
Disperse Orange 59 EKT, ICC.
Disperse Orange 62-- BUC, DUP.
Disperse Orange 65 VPC.
Disperse Orange 75 DUP.
Disperse Orange 78 TRC.
Disperse Oiange 89- AAP.
Disperse Orange 90 . AAP.
Disperse Orange S4--« SDC.
Other disperse orange dyes AAP, ALT, ATL, EkT, GAP, MAY, SUC, VPC.
•Disperse red dyes:
•Disperse Ited I -'- AAP, DUP, DKT, GAP, UN, HSH, ICC, TRC.
Disperse Ked 4 -- GAP, ICC, TRC,
•Disperse Red 5 - '--- AAP, EKT, GAP, HSH, ICC.
Disperse Red 7 * AAP, GAP.
Disperse Red 9 . ATL.
•Disperse Red 11 -- AAP, DUP, GAP, ICC.
•Disperse Red 13 - -'- _ AAP, DUP, GAP, ICC.
•Disperse Red 15 - GAP, HSH, ICC, TRC.
•Disperse Red 17 1 _ AAP> Dup> EKT) GAF( ICC( TRC
Disperse Red 30— - - EKT, TRC.
Disperse Red 31 1 ... . ICC.
Disperse Red 35 * EKT!
Disperse Red S4 . ICC.
•Disperse Red 55-- - AAP, DUP, CAP, HN, TRC.
Disperse Red 56 ' OUP
Disperse Red 59 - ACYJ DUP, GAP.
•Disperse Red 60—-* - AAP. ALT. ATL, DUP, EKT, GAP, HN, SDC. TRC,
•Disperse Red 65 -'- DUP) EKT> ICc! TRC;
• Disperse Red 66 AAP.
Disperse Red 73 '. TRC.
Disperse Red 78 ICC TRC
Disperse Red 82 VPc!
Disperse Red 86 EKT GAP.
Disperse Red 88 EKT!
Disperse Red 90 VPC
Disperse Red 96 ACY!
Disperse Red 117 EKT.
Disperse Red 133-
Disperse Red 136-
' Disperse Red 137-
Disperse Red 138-
Dispersc Red 140-
Disperse Red 159 VPC
Disperse Red 161-
Dispcrsc Red 167-
Disperse Red 176-
Disperse Red 177-
Disperse Red 178-
Disperse Red 179-
Disperie Red 180 . jcc
•Disperse Violet'1 - Mp w HSH 1 ' * ' ILL' 1KC>
Disperse Violet 41-
Dispcrse Violet .12 EKT
Diiperbc; Violet 43
Disperse Violet 44
Other disperse violet dyes CAP j^y SIIC
265
-------
TABLE 4.--DYES FOR WHICH II.S, PRODUCTION OR SALES WERE: REPORTED,
IDENTIFIED BY MANUFACTURER, 1971—CONTINUED
Dye
DISPF.RSE DYES— Continued
'Disperse blue dyes:
'
i^perse uc
P
1 1)L c
ispeibe ue
n • u i i T
*SPel" U1l C ,77
isperse ue ,
Disperse brown Jyes:
'nispcrso bl.K'k dyes, 9
mr.R-Hrvnvr iwus
'Reactive \flloK ilycs:
Manufacturers,' iJcnt i ticat ton codes
^according to list in table 31
AAP, BAS, GAP, ICC, TPC.
A-\P, DUP, HKT, GAP, UN. HSU, ICC, 1 liC .
tKT, GAI;, HN, HSU, ICC, TUC .
&\F, ICC.
EKT, TRC.
ICI.
ICC,
TRC.
VPC.
TRC.
DUP.
DUP.
DUP, GAP, SI/C.
DUP.
DUP, UKT, GAP, TRC.
AAP.
VPC.
ICI. -
IRC.
AAP, EKT, TRC.
Vf'C.
TRC.
BAS.
GAP.
EKT.
DUP.
EKT.
EKT.
EKT.
faKT .
EKT, GAI'.
EKT.
EKr.
TRC.
DUP.
VPC.
DUP.
HST.
UAF.
ICC.
AIT, ATI., DUP, fkT, CAP, HN, HSH, ICC, MAY, SI1C
TRC, VPC.
CAK, TRC, VPC.
TRC.
CUP, EKT, GAP.
CKT.
VPC .
AAP.
GM-, ICC, SDC.
AA?, WP, GAP, IRC.
\1l, TkC.
AAP, I.KT.
tKT.
LKl1.
AIT, ATL, iiiji1, u\^, icc, snc, VPC.
usr, u i .
i HI: .
1 PC .
H'-r, in.
266
-------
TABLE
,— DYES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED,
IDENTIFIED BY MANUFACTURER/ 1971--CONT! NUED
Dye
Manufacturers' iJcnt i fic.it ion codes
(according to libt in t.ihlc S)
FIBER-REACTIVE DYES--Continued
•Reactive yellow dyes--Continued
Reactive Yellow 6 HST, TRC.
Reactive Yellow 7 -- HST, ICI.
Reactive Yellow 13 HST.
Reactive Yellow 18 --- ICI.
Reactive Yellow 22 ICI.
Reactive Yellow 25 - VPC.
Reactive Yellow 31 HST.
Reactive Yellow 37 [1ST.
Reactive Yellow 60 - ACY.
Reactive Yellow 61 ACY.
Reactive Yellow 62 - - ACY.
Other reactive yellow dyes HST.
Reactive orange dyes: t
Reactive Orange 1 ICI.
Reactive Orange 4 ICI.
Reactive Orange 5 TRC.
Reactive Orange 12 ICI.
Reactive Orange 13 ICI.
Reactive Orange 14 ICI.
Reactive Orange 16 HST.
Reactive Orange 50 HST.
Other reactive orange dyes HST.
Reactive rud dyes:
Reactive Red 1 ICI.
Reactive Red 2 -- ICI.
Reactive Red 4--- --*- TRC.
Reactive Red 5 ICI.
Reactive Red 8 -, * -- ICI.
Rejctive Red 11 -*- ICI, TRC.
Reactive Red 21 - HST.
Reactive Red 29 ICI.
Reactive Red 31-- -- ICI.
Reactive Red 33 ICJ.
React: c .
-------
TABI.E4.~Dyr: ;; .
Dyo Miuuf.icturers ' iclcnt ificat Jen codes
( (aCLoruiiig to lisi in t.l.U- .-, )
nur;.-pr.\ci IVT r>Yhs--rontuu!cd
"Kjactive him1 Jver - -Coi'tir.iir-il
KLMO 1 1 ' e gr L rp ' us.
,CaC 1V,^ /' ' ' " -.,
R(-Mi-ti\e bro^n .jv e1. .
n )C, -VVt -' °W' (' ,
'Reactive bl.ic1- Jvi s
FLHOULSa-M URIC
Fluorescent Brightening X ;e!it
riu'iruscent Brig',i!.i nun; i\, ciit
Fluore.,ccnt Bright rn in,; ^;;t--it
Fluorescent Brightening Atjeiit
f'luoro?cerit fin ;'iucni rig At'ciit
l'luore>cent lirigtuenin:; \t'.i;nt
Fluorescent. Brightening ng'.':it
1-luorescoiit Bt -,>;ht cmr.g Aftent
1 luorescent Briynti ni r.g Aj.jnt
Tluorescent Brifjiteiuiig Ajji'iit
i luorescent Brightening '\^ent
Fluorescent bri^hti-ning Aj;ent
. luorescent 'Irightcn.nff A^rat
fluorescent Cri^htemnK Av.snt
Fluorescent bright ciun" Aijent
riuoresoent Bviijlit oiling -\fent
Fluorescent Briniitcniiig Agent
Fiuore^ccnt lirLghteninii Agent
Huoresccnt Bj-i^ri toning A^ent
Iluorosc«nt Ih ightening Ap.ent
rluorcsccnt brightening A"ent
.'luorescent Brightening Agent
Huorescc-nt Hrighteninn A;unt
fluorescent 'iri.-litening Agent
Fluorescent Brightc-ning ^penc
Flijoicscent Bn^htrring Agent
1 luorescent BTightei'in^; Agent
t\a\ fit ^resci^nt hrij'lucning
FOTJD, DKUC, AND
Fcod, ''i'U:j, arvl
" '-O1"' Blue No 2 .„_-_
"M'*L Red No ^ -
• • r v v
^
HI IN ING AGtNTS
J
6-
24 -
V)
- .,
4f,
52
54 _„ _^__
r,9
(,^ _ _ _ ._
7 j^
IQg _ _ .
1(J9 -- -- _.-
1 ^S
U6 1 - ---
1 *iR -
J3Q . _^ _
1^4 ^
|T;n _ _ _^_,
r"
COSMI VIC COLORS
.'', M.T,-ti." /)!/CO
•
/
•
•
.Ki-
II. 1 .
l.'.i , 1CI .
1.1.
1 1ST.
l.i.
r , inc.
ACV.
(M~ , SDil.
Cd". .
i.GY.
/C-i, CCK, DUP, SDH, VPC.
C,AI\
r\i:.
'• RC
LG,'.
\CY .
(.CW, GAP.
:CY, GAF.
C.M-.
D1J!', Vl'C.
L.AF.
ACY.
. 1)11.
VC. ( .
V ', CCW, CCY, GAF, rChf, S, Vl'C.
' ,, AIT, KON, SUM, KJ.
,. ., AI.J', kO.N, .Sl'll, WJ .
r, -i i .
•:, ALT, ICON, SDH, STC, h.J .
., Al i , kON, Mill, Slli, n.l .
. , KON, Bl'G.
,. [ill, U.
.1 i \! r i os sic wj
u I, .M.I , kO'(, M'll, SI'G, W 1 .
268
-------
TABLE4t--DvES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED,
IDENTIFIED BY MANUFACTURER. 1971—CONTINUED
Dye
Manufacturers' identification codp5
(according to list in table 3)
FOOD. DRUG, AND COSMETIC COLORS— Continued
Drug and Coemetic Dyes
D&C
05C
DSC
DSC
»D£,C
DSC
DSC
DSC
DSC
DSC
*D5C
*Df,C
DSC
*D6C
D6C
DSC
D5C
DSC
Df,C
•DSC
•DSC
DSC
D6C
D5C
D5C
D&C
*D5C
DSC
DfiC
DSC
D6C
DSC
DSC
DSC
Blue No. 6-
Green No. 5
Green No. 6
Green No. 8
Orange No.
Orange No.
Orange No.
Orange No.
Red No. 2--
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Red No.
Yellow
Yellow
Yellow
Yellow
Yellow No.
Yellow No.
3--
6--
7--
8--
9- -
10-
11-
1J-
17-
19-
21-
22-
27-
28-
30-
31-
33-
. 34-
. 36-
. 37-
No.
No.
No.
No.
Drug and Cosmetic Dyes, External
Ext. DSC Green No. 1--
Ext. DSC Yellow No. 1-
Ext. D5C Yellow No. 7-
INGRAIN DYES
Ingrain blue dyes:
Ingrain Blue 1 —
Ingrain Blue 3
MORDANT DYES
•Mordant yellow dyes:
•Mordant 'ellow 1 --
Mordant Vellou '
Mordant Yellow :>
•Mordant Yellou 8
Mordant Yellow 14-
Mordcint Yelloh 16- •
Mordant Yellow TO---
\lordoiit Yellow 76- --
Mordant Yellow 29
Mordant Yellow TO---
KON.
ACS, ALT,
ACS, ALT,
KON, SDH.
KON, SNA,
SNA, TMS.
IMS.
SNA.
KON.
KON.
KON, SNA,
KON, SNA,
KON, SNA.
KON, SNA,
KON, SNA.
KON, SNA.
SNA, TMS.
SNA, TMS.
KON.
ACS, KON,
KON, SNA,
KON.
SDH, SNA,
ACS, TMS.
KON, TMS.
KON.
ACS, KON.
KON.
ALT, KON,
ACS.
KON, TMS.
KON.
ALT, KON.
KON, TMS.
KON.
KON.
ACS, KON.
ACS, KON.
KON.
ICI.
ICI.
ATL, GAP,
ATL.
TRC.
ACS, PDC,
ACS.
ACY.
ACS, ATL.
VPC.
C.AF .
TRC, VPC.
KCN.
KON.
TMS.
TMS.
TMS.
TMS.
SNA, TMS
TMS.
TMS
TMS.
PDC.
VPC.
-------
TABLE 4.—DYES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED,
IDENTIFIED BY MANUFACTURER, 1971—CONTINUED
Dye
MORDANT DYES--Continued
*Morda it orange dyes :
' ' ° *
M T- ' n ' a *
*M'jrdani r^J dyes:
Mordant violet dyes:
Mordant blue dyes:
*
Mordant green dyes:
''iordant brown dyes:
«
*Mordant black dyes:
OXIDATION BASES
SOLVENT DYES
•Solvent yellow Jyos: f
Manufacturers' identification coJc'..
(according to list in table 3)
ACY, PDC, TRC.
GAP.
ATL, GAT, PDC, TRC.
TRC.
ACY.
PDC.
ACY, ATL, EDO, CMC, GAP, I'DC, TRC, VPC .
MRX.
ACY.
PDC.
PDC.
GAP.
GAP.
GAP.
GAP.
ACS.
CMC.
ACY.
PDC.
ACS, CMC, DUP, GAP, TRC, YAW.
PDC.
ACS.
GAP.
ACS, DUP.
GAP.
GAP, VPC.
ACS, GAP, PDC, TRC.
ACS, CMC, GAP, VPC, YAW.
TRC.
TRC.
DUP, PDC.
ACS.
ACS, TRC.
GAP.
VPC.
ACS. VPC.
ACS, GAP, TRC, VPC.
HSU.
ACS, ACY, GAP, TRC.
PDC.
TRC.
•
ACY.
PDC.
ACY.
ACY.
ACY, CMC.
AAP.
AAP, DUF, GAP, PSC.
ACS, PSC.
ACY, GAP, PSC.
270
-------
TABLE 4---DYES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED,
IDENTIFIED BY MANUFACTURER, 1971—CONTINUED
Dye
Manufacturers' identification codes
(according to list in table 3)
SOLVFNT DYES--Continued
•Solvent yellow dye^-Continued
•Solvent Yellow 1-1
Solvent Yellow 19
Solvent Yellow J9-
30-
Solvent Yellow
Solvent Yellow 3.<
Solvent Yellow 34
H»!vent Yellow 40 --
Solvent Yellow 42
Solvent Yellow 4? - —
Solvent Yellow 44
Solvent Yellow 45-
'Solvent Yellow 47
Solvent Yellow 56
Solvent Yellow 71
Solvent Yellow 7?
Solvent Yellow 87
Other solvent yellow dyes--
*Solvent orange dyes:
Solvent Orange 2
•Solvent Orange 3 -
Solvent Orange S--
•Solvent Orange 7
Solvent Orange 20
Solvent Orange 23
Solvent Orange 24---
Solvent Orange 25---
Solvent Orange 31 *
Solvent Orange 48-- —
Solvent Orange 51--
Other solvent orange dyes-
*So}vent red dyes:
Solvent Red 1
Solvent tted S
Solvent Red 22
Solvent Red 24
•Solvent Red 26
Solvent Red 27
Solvent Red 33--
Solvent Red 35
Solvent Red 40
Solvent Red 41
•Solvent Red 49
Solvent Red 52
Solvent Red 68
Solvent Red 09
Solvent Red 74
Solvent Red 75-
Solvent Red 105
Solvent Red 108 •- -
Solvent Red 111
Solvent Rod 115- --
Solvent Rod 126--
Other solvent red dyes— -
•Solvent violet Jyes:
Solvent Violet 8 - - --
Solvent Violet 9- -- --- - -•
Solvent Violet 13
Solvent Violet 14
Other solvent violet dyes--
Solvent blue dyes'
Solvent lilue 3 -
Solvent Blue -5
Solvent Blue 5
AAP,
GAP.
GAT.
ACS.
AAP,
ACY,
ACS.
ACS.
GAP.
ACS,
ACS.
ACY,
ACS,
ACY.
ACY.
ACY.
AAP.
AAP,
ACS,
GAP.
ACS,
ACY,
ACS.
DUP.
ACY,
ACS.
ACY.
ACY.
AAP,
PSC.
GAF.
GAP.
ACY,
AAP,
ACS.
DUP,
GAF.
GAf .
DSC.
ACY,
AAP,
ACS.
DSC,
ACS.
ACS.
ACY.
ACY.
ACY.
ACY.
ACY.
AAP
ACS, ACY, DUP, GAP, PEC
ACS, ACY.
DSC.
GAF.
DUP, GAF.
ACY.
ATL, DSC, HAT.
PSC.
ACY, DSC, GAF, PSC.
ACY, GAF.
GAF.
DUP.
ACY, DSC, DUP, PAT.
DUP, GAF.
ACS, ACY, PSC.
GAF.
DSC, DUP, GAF.
GAF, ICI.
DUP.
ACY, ATL, DSC. PUP, ICI .
ATY.
DSC
UP,
AAP,
AAP,
ACY,
DSC,
D.SC.
DSC.
ATL, HSU. ICI.
ICI.
DSC, PAT.
SW.
UUP, SDH.
271
-------
TABLE 4,—DYES FOR WHICH U.S. PRODUCTION OR SALES WERE REPORTED,
IDENTIFIED BY MANUFACTURER, 1971--CONTINUED
D> s
SOf,VLNT PYLS— Continued
Solvent blue dyes -Continued
So Iv fill t;reen dyes :
, Vl"n' ")' "'"'" t,
01 ' '-'" l IL( ' ""*
Solvent 1 * ^-j elves : ^
Solveni. bJdds Jyos:
Manufacturers' identificat ion codes
(according to list in table 3)
•
DSC
ACY
GAP
EDO GAP ICI
EDO
ACS '
ACS DUP
DUP
ACS ACY ATL DUP GAP
ACS
DUP
ACY
AAP ACY DSC GAP ICI PAT x
*
ACY DSC
G\F
GAP
ACY D SC G AF
D'fP '
DUP PSC
APV *
DSC
ACS
DUP
272
-------
Table 5.
Acid Yellow 77
Orange 4
Red 42
Red 100
Blue 10
Blue 89
Blue 161
Black 138
Basic Yellow 26
Red 23
Direct Yellow 9
Red 5
Red 46
Violet I
Blue 238
Green 46
Brown 3
Brown 33
Azo Dyes Produced in 1971 but Not in 1972
ACY
ACY
GAF
VPC
AAP.ACS
ACS
VPC
VPC
ACY
VPC
ATL
ACS
ATL
ACS, ATL
ACY
VPC
VPC
duP
Disperse Orange
Orange
Red
Reactive Blue
Blue
Black
Mordant Yellow
Red
Blue
Green
Brown
Black
Black
13
28
56
9
20
1
3
5
13
11
50
1
7
HST
AAP
duP
ICI
HST
HST.TRC
ATL
PDC
ACS
ACY
TRC
ACS
GAF
273
-------
Table 6. Azo Dyes Produced in 1972 but Not In 1971
Acid Yellow 4 SDH Reactive Yellow 15 HST
Yellow 17 HST
Black 5 1ST
Red 40 ACS.WJ
Red 39 SDH
Disperse Red 21 EKT Mordant Yellow 36 PDC
Yellow
Orange
Red
Red
Black
Black
4
5
179
277
53
139
SDH
ACY
TRC
VPC
PSC
VPC
Reac
FD&C
D&C
27k
-------
TABLE 7.—DYES! DIRECTORY OF MANUFACTURERS,, 1971
ALPHABETICAL 'DIRECTORY BY CODE
(Names of dye manufacturers that reported production or sales to the U.S. Tariff Conaisslon for 1971 are listed
in the order of their identification codes a* used in table 2]
Code
Name of company-
Code
Name of company
AAP
ACS
ACY
ALL
ALT
ATL
BAS
BDO
BUG
CCW
CGY
ore
CPC
ow
DSC
DUP
ECT
FAB
GAF
KN
HSC
HSH
HST
American Aniline Products, Inc.
Allied Chemical Corp., Specialty Chemicals
Div.
American Cyanamid Co.
Alliance Oienical, Inc.
Cronpton S Knowles Corp., Althouse Div,
Atlantic Chemical Corp.
BASF Wyandotte Corp.
Benzenoid Organic*, Inc.
Blackman-Uhler Chemical Co.
Cincinnati Malacron Chemicals, Inc.
Ciba-Geigy Corp.
Nyanza, Inc.
Childs Pulp Colors, Inc.
Upjohn Co., Fine Chemical Div.
Pye Specialties, Inc.
E. I. duPont de Nemours & Co., Inc.
Eastman Kodak Co., Tennessee Eastusn
Co., Div.
Fabricolor Manufacturing Corp.
GAF Corp., Chemical Div,
Tenneco Qiemicals, Inc.
Chemetron Corp., Pigments Div.
Harshaw Cheaical Co. Div. of Kewsnee
Oil Co.
AoericsJi Hoechst Corp.
ICC
ICI
RON
HAY
MRX
PAT
PC*
PDC
PSC
S
SDC
SDH
SNA
STC
ST6
SK
TRC
we
YAK
Innont Corp.
ICI America, Inc
H. Kohnstanun & Co., Inc.
Otto B. May, Inc.
Max Marx Color S Chemical Co
Morton International, Inc., Morton Oienical
Co. Div.
Pfister Oienical Works
Berncolors-PougJjkeepsie, Inc.
Passaic Color § Chenical Co,
Inc., Sandoz Color 5 Chemicals Div.
Mtrttn-Marietta Corp., Southern Dyestuff
Co. Div,
Sterling Drug, Inc., Hilton-Davis Chemical
Co, Div,
Swi Oienical Corp.
Sou-Tex Chemical Co.,
Inc,
Stange Co.
Sherwin-Williams Co.
Sterling Drug, Inc., Thomasset Colors Div.
Tons River Chemical Corp.
Verona Corp.
Warner-Jenkinsnn Manufacturing Co.
Y.S. Young, Youig Aniline Works Div.
hlote."--Ccaiplete~naiaes'"and address of* the"'above reporti'nf coapanies, will be listed in the Tariff CommissTonTs~
annual report, Synthetic Organic .ChenicalSj aJnited States Production and Sales, 1.971.
275
-------
BROMINATED HYDROCARBONS
SUMMARY AND CONCLUSION AS TO DEGREE OF HAZARD
Only two bromohydrocarbons, methyl bromide and ethylene dibromide,
are known to be in large scale production and use. Most of the methyl
bromide is used in the fumigation of stored agricultural products and
soil sterilization at an annual increase of about 10%. Most of the
ethylene dibromide is used in leaded gasoline as a scavenger for lead
deposits in engines; perhaps an amount equivalent to that of methyl bromide
is used for the same purposes. Production is expected to decrease with
the decreased usage of leaded gas.
Both react to some extent, especially the methyl bromide, with pro-
tein in the foods they contact. Resultant toxicity or reduction in nutri-
tional values is thought to be Oi'little concern to humans or animals fed
foodstuffs which have been given sufficient time to allow residual, un-
reacted fumigant to evaporate. Egg size and quantity may be reduced in
poultry fed too much ethylene dibromide as residue in the feed.
A variety of metabolites comprises the urinary pathway of excretion
of bromohydrocarbons. These include alkyl and hydroxyalkyl mercapturic
acids and S-oxide mercapturic acids, similarly changed peptides, etc.
Complete breakdown to carbon dioxide has also been demonstrated.
Methyl bromide is a very toxic substance with many known fatalities
from occupational use. Its low detectability by human senses at fatal
air concentrations is especially dangerous to dock and warehouse workers
who may not have been informed that a cargo or shipment was recently fumi-
gated. Death from an acute dose usually results from lung damage, but
kidney damage is also immediate. Chronic exposure can produce brain and
spinal cord damage, occasionally with effects lasting long after cessation
of exposure.
276
-------
Ethylene dibromide is rated as a highly toxic substance, but its
relatively low volatility is probably the contributing factor in a lack
of reported fatalities connected with its use.
Neither compound appears to offer an environmental threat, there
being no indication of accumulation in laboratory animals given extra-
ordinary doses, and there being no indication of effective soil accumula-
tion from annual application.
277
-------
BROMINATED HYDROCAEBONS
I. PHYSICAL PROPERTIES
Some appropriate physical properties of many of the C^-Cs mono-
and polybromohydrocarbons are given in Table 1. The references used,
Dow Chemical and Sax, provide similar information on many other bromo-
hydrocarbons of considerably lesser economic or toxicological importance
but frequently used in research laboratories. Tht_ compounds in the
table should be considered soluble in a variety of organic solvents and,
at best, slightly soluble in water. Tetrabromomethane, ally! bromide
and propargyl bromide are lachrymatory. The vapor pressure of ethyl
bromide is 400 mm Hg at 21°C, that of ethylene dibromide is 17.4 mm Hg
at 30°C, and that of methyl bromide is 1420 mm Hg at 20°C.
Hassall (1953) reported the following saturation vapor pressures
at 25° in mm Hg: ethyl bromide, 468; propyl bromide, 135; butyl
bromide, 38.9; amyl bromide, 13.8; hexyl bromide, 10.
Saracco and Marchetti (1958) provided the following equation for
estimating the water solubility of bromohydrocarbons (straight chain):
InS = lnSo - Kn, in which S is in units of moles/liter, So has the
value 1.63, K has the value 1.46, and n is the number of carbon atoms
in the chain.
Hill (1962) studied the explosive limits range of air-methyl
bromide mixtures as a function of pressure, and found that increases
in pressure over atmospheric allowed mixtures relatively rich in methyl
bromide to explode. For example his range at one atmosphere was 10-15.4%
methyl bromide, but at 8-9 atmospheres a 23% methyl bromide mix ex-
ploded, and at 6-7 atmospheres a 29.5% mix exploded.
278
-------
Table 1. Properties of Some Bromohydrocarbons"
Freezing
Point
Methyl Brona.de
CH3Br -94.1
Methylene Dibromide
CH2Br2 -52
Bromoform
CHBr3 7.8
Te t Tab romome thane
CBrit ' 92-3
Ethyl Bromide
GH3CH2Br -119.3
Vinyl Bromide
CH2=CHBr -139.2
Ethylene Dibromide
BrCH2CH2Br 10
Acetylene Tetrabromide
Br2CHCHBr2 -0.1
Propyl Bromide
CH3CH2CH2Br -109.9
Allyl Bromide
GH2=GHCH2Br <-50
Propargyl Bromide
CHgCCH2Br -62
a - from Dow Chemical Co. Bulletin
b - in degrees centigrade
c - from Sax
d - at 25/25°
Boiling Flash Fire , Autoignition Expl. Sp . Gravity,
Pointb Pointb Point Pointb'c Limitsc Vapor density
3.6
99
148.9
189
38.4
15.8
131.4
245.8
(dec.)
71
70.2 -1.1 32.28
84.4 21.1 expl.
164-100-68 (1968)
536 10-16% 1.746(-5)f,
3.27
2.49
2,88
511 6.7-11.3% 1,4492,
3.76
1.549(10)f
2'. 17,
6.48
2.96
1.350
1.412
1.582
e - at 25°
f - density at (x°C)
g • open cvi
- none
Index of
Refraction0
1.5381
1.5944
1.4210
1.4412(10)
1.5360
1.6350
1.4314
1.465
1.4912
From Dangerous Properties of Industrial Materials by N.I. Sax c 1975, 1963 oy Litton
Educations Publishing, Inc. Reprinted by permission of Van Nostrand Reinhold Company.
-------
Forshey et al (1969) studied the fire and explosion potential of
propargyl bromide. Vapors would propagate a flame in a 19-cm
diameter container at a gauge pressure of 0.03 psia at room temperature.
Accidental pressurization of the aerated liquid could ignite it.
II. PRODUCTION
The U.S. Tariff Commission Reports contained the following figures
for production (in metric tons):
Methyl Bromide Ethyl Bromide Ethylene Dibromide
9,080
1969
1970
1971
1972
1973
791
9,540
140,600
134,800
127,000
143,100
11,160
13,410
The April 1974 U.S.T.C. Preliminary Report on 1972 Miscellaneous
Chemicals production listed the following brominated hydrocarbons and
manufacturers:
Michigan Chemical Corp. (MCH)
Abbott Labs., Eastman Kodak Co. (EK)
1-bromobutane
2-bromobutane
b romoe thane
1-b romohexane
1-b romo-3-methyl-butane
l-bromo-3-methyl-2-
butene
1-b romo-oc ta decane
1-bromo-octane
Dow Chemical Co., Great Lakes Chemical
Corp. (GTL), MCH
Humphrey Chemical Co.
Eli Lilly & Co. (LIL)
Sterling Drug, Inc. - Winthrop Labs.
Div. (SOW)
du Pont
MCH
280
-------
2-b romopentane
1-b romopropane
1,2-dib romoe thane
LIL
EK, SDW
Dow, GTL, MCH, Pittsburgh Plate
Glass Co., Ethyl Corp.
Dibromomethane Dow
In the June 1974 Preliminary Report on Pesticides and Related Products
for 1973 the following manufacturers are listed for methyl bromide:
Kerr-McGee Chemical Corp., Dow, GTL, and MCH.
The Chemical Week Buyers Guide for 1974 lists the following
compounds for sale:
hexamethylene dibromide
n-hexyl bromide
methylene dibromide
nonyl bromide
octadecyl bromide
octyl bromide
pentamethylene bromide
propylene dibromide
tetrabromomethane
tetradecyl bromide
t rime thy lene dibromide
undecyl bromide
vinyl bromide
Product bulletins from Dow and White Chemical Corporation indicate
that both can supply a wide variety of bromohydrocarbons.
acetylene tetrabromide
allyl bromide
n-amyl bromide
i-amyl bromide
bromocyclohexane
bromocyclopentane
bromoform
i-butyl bromide
s-butyl bromide
t-butyl bromide
n-decyl bromide
n-heptyl bromide
n-hexadecyl bromide
281
-------
III. USE
Ethylene dibromide, the largest tonnage bromohydrocarbon, is used
mostly as a lead scavenger in leaded gasoline. It is difficult to
project a future trend in this area because of the possibility of
changes in the consumption of tetraalkyl lead in gasoline in connection
with exhaust emission regulations and potential engine design changes.
While it is likely that the new engines will use unleaded or lightly
leaded gas, there will remain in existence for years millions of cars
intended to be fueled with high lead gas. A relatively small amount of
production is used as a fumigant for stored grain, for soil, as a dye
and pharmaceutical intermediate, and as a solvent.
Methyl bromide has been used in the past as a fire extinguishing
agent, under the tradename Halon 1001. Its extremely high toxicity,
coupled with its tendency to corrode the usual metallic containers,
ended this use. Petrella and Sellers (1970) compared Halon 1001 with
a number of other Halons (mixed-halogen compounds of methane or
ethane) in their relative fire extinguishing capabilities; their con-
clusion was that the toxicity of Halon 1001 far outweighed its super-
iority.
Most of the methyl bromide produced is used as a fumigant for
stored agricultural products and as a sterilizing agent for Eioil. Its
high volatility requires that an enclosure or impermeable cover be
present to ensure complete and economic utilization. When used in
buildings it is blended with 2% of chloropicrin (CC13N02), the
lachrymatory action of which acts as a warning for the methyl bromide
which is undetectable by human senses in deadly concentrations.
282
-------
The major use for ethyl bromide, and a minor one for methyl
bromide, is as an alkylating agent in drug manufacture. Lesser
amounts of the ethyl are used as a solvent or refrigerant.
The following table on properties and uses of other bromohydro-
carbons was adapted from one in Kirk-Othmer, Vol. 3, pp 776-8 (1964).
Table 2. Properties and Uses of Miscellaneous Bromohydrocarbonsc
Compound Mp, °C Bp, °C
Acetylene tetrabromide
Allyl bromide
Bromoform
n-Butyl bromide
CH3(CH2)2CH2Br -112.7 100.5
Carbon tetrabromide
Ethylidene bromide
CH3CHBr2
Isopropyl bromide
(CH3)2CHBr -89.0
Lauryl bromide
CH3(CH2)10CH2Br
Methylene bromide
Propargyl bromide
n-Propyl bromide
CH3CH2CH2Br -110 70.9
Propylene bromide
CH3GHBrCH2Br -55.3 140
1,2,3-Tribromopropane
BrCH2CHBrCH2Br 16 220
Trimethylene bromide
BrCH2CH2CH2Br -34.2 167.3
Vinyl bromide
d(20/4°)
20
Useb
G,H,M,
Solv
F, Syn
G.H.P,
Syn
1.2687(25/4°) 1.4398 Syn
3.42
109
59.3
177
(45 mm)
2.06
1 .3138
1.0382
L.3514
1 .9333
2.4076
(25/4°)
1.9790
1.60 BrominaU I.K
(99.5) agent
1.5122 H, Syn
1.4254 Solv, Syn
].4581 Syn
E,G,H,
Solv, S>r,
F, Syn
1.4341 Solv, ay,,
1.5194 Solv, Syn
1.5835 H, Syn
(25)
1.5232 Syn of
cyclopropane
Copolymer
a - Properties of those compounds also in Table 1 are not reproduced
b - Explanation of letters and abbreviations is as follows:
(Table 2 reprinted with permission from Kirk-Othmer Encyclopedia,
Vol. 3, pages 776-778 (1968). Copyright by John Wiley & Sons -
Interscience Publishers.)
283
-------
E - Ingredient of fire-extinguishing fluids or as a fire retardant
F - Fumigant, if very volatile, or contact poison
G - Gage fluid
H - Heavy liquid for flotation-type ore separation
M - Microscopic or refractometric fluid
P - Ingredient of medicinal or pharmaceutical products
Solv - Solvent, generally for fats, waxes, or resins, possibly
as a reaction medium
Syn - Intermediate in synthesis of other compounds
Barduhn et al (1960) examined methyl bromide and found it to be
promising as a demineralizing agent for sea water because of the
hydrate it forms under pressure;.
Huang et al (1966) studied the concentration of fruit juices by
the use of methyl bromide to remove some of the water as complexed
crystals. While an effective concentration was achieved, some of the
natural flavor was lost and an undesirable flavor added.
IV. CURRENT PRACTICE
ICC shipping regulations for liquid methyl bromide require poison
and poison B labels, and limit the quantity to 208 liters (55 gal.) The
Coast Guard requires poiaon, poison B, and MCA warning labels. The
IATA does not allow it on passenger craft, but does allow 220 liters (58 gal.)
on cargo craft with poison and poison B lables.
General regulations for ethyl bromide call for the MCA label; IATA
gives it a Class A status, allowing 40 liters (10.6 gal.) on passenger^
220 liters (58 gals.) cargo crafts.
28U
-------
Ethylene dibromlde must have the MCA label. It is required by
IATA to have the poison and poison B labels, and is limited to one liter
on passenger and 220 liters on cargo craft
IATA requires butyl bromide to bear the Red label, and limits it
to one liter on passenger, 40 liters on cargo craft.
Allyl and propargyl bromides require flammable gas labels.
V. ENVIRONMENTAL CONTAMINATION
Presumably a large amount of methyl bromide is released into the
air as normal operating procedure, as the products it is used to
fumigate must be aerated before being consumed or processed further.
Likewise soil sterilized by methyl bromide injection must be aired to
minimize damage to seeds or seedlings.
A similar situation exists with the fumigating uses of ethylene
dibromide. The latter has another route into the air by way of unburned
gasoline from auto exhausts. No reports dealing with the extent of
these emissions or effect on them of emission controls were found.
Leonard and Lider (1960) injected ethylene dibromide into soil
and found that lateral diffusion was limited. This may mean that
accidental spills onto soil in populated areas could be counteracted
by prompt removal of the soil to a safer area.
The use, as indicated in Table 2, of certain bromohydrocarbons
as flotation agents would release fumes into the air, and probably some
liquid is trapped in any discarded materials.
2H5
-------
VI. MONITORING AND ANALYSIS
Wade (1952) described a colorimetric analysis for bromide ion ob-
tained from air samples containing bromohydrocarbons - not applicable
if chloro- or bromochlorohydrocarbons are present. In a standard
technique for sampling for this type of contamination, the air sample
is drawn through a catalytic furnace into a bubbler containing alkaline
hydrogen peroxide. This solution is transferred to a beaker and boiled
down to 2-3 ml. After transfer to a calibrated test tube, and acidi-
fication with sulfuric acid, a fixed amount of aqueous NaAuCl^ is
added. The color developed is read against a reagent/treatment blank
at 470 nm and compared with a calibration chart. No changes are re-
quired for the range 0.1-4.0 mg of bromide ion. Accuracy is less than
silver nitrate titration, and better approximates the latter at the
lower end of the range.
Lugg (1955) described a colorimetric method for determining a
minimum of 50 mg/cu. m. (13 ppm) of methyl bromide in air. A 10-1
sample of air is drawn through a one-1 Winchester bottle. Add 15 ml
of distilled pyridine containing 1% water; stopper and wet the bottle
sides with the pyridine. Let stand seven hours, shake, invert, and
let drain. To 10 ml of the solution in a 2.5 x 15-cm test tube, add
1/2 ml of 0.5N NaOH and heat for 15 minutes at 95°C. Cool five
minutes in an ambient water bath. Add 1/2 ml of 2% aqueous India gum
and read in a colorimeter within 10 minutes against water over the
range 370-430 nm. There is a linear relationship between absorbance
and amount of methyl bromide over the range 0-1.5 mg, but the minimum
recommended is 0.1 mg. Ethylene dibromide interferes but not: seriously,
likewise ethyl chloride and ethylene dichloride; serious interference
would come from methyl and ethyl iodides, and ethyl bromide.
286
-------
Heseltine (1959) described a commercially available detector tube
for methyl bromide which was suitable for monitoring fumigated
enclosures.
Smith and Shigenaga (1961) described a technique for extraction
of sterilants from soil. A 25 g soil sample, wetted with 20 ml of
water, was shaken for 30 minutes with 2 ml of n-hexane for ethylene
dibromide or o-xylene for propargyl bromide. A 5-50 \j,l aliquot of the
extractant was then injected directly onto a gas chromatographic
column. Recoveries ranged over 80-90%.
Alon et al (1962) analyzed for residual acetylene tetrabromide in
ore from flotation processing, and in the hydrocarbon used to recover
the bromohydrocarbon. Alcoholic KOH was used to remove the bromide as
HBr. Oxidation to bromate could be followed by an iodometric finish
when the sample contained 400 yg-10 mg of bromide in the presence of
chloride, or by a colorimetric finish based on formation of tetra-
bromorosaniline, in the 0-20 yg bromide range. For the 10-200 mg
bromide range, in the absence of chloride, an argentometric method was
used, and for production control, the nephelometric metliod as silver
bromide.
Dumas and Latimer (1962) analyzed atmospheric methyl bromide
using f 35-ml samples containing 1-100 mg/1 of methyl bromide. After
drawing the sample into an evacuated flask, 0.5 ml of IN potassium
hydroxide was added and heated at 60°C for 45 minutes. Excess alkali
was neutralized with IN nitric acid. Then the sample was titrated with
a modified Fisher Coulomatic Titrator.
Dumas (1962) analyzed atmospheric ethylene dibromide by drawing a
28?
-------
sample into an evacuated flask, adding 1 ml of 0.5N sodium hydroxide,
and refluxing for 15 minutes to remove one of the bromides. This was
quantitized on a Fisher Coulomatic Titrator. Results were good for
the 0.75-30 mg range.
Lindgren et al (1962) analyzed grain for residual combined
bromide and methyl bromide after fumigation by neutron activation
analysis. A 5-gm sample was irradiated for 30 minutes at a flux of
1.8 x 10*2 neutrons/sq. cm. sec at a power level of 250 kW. The
Br-82 0.77-Mev gamma ray intensity was measured after a 2-4 day decay
period and compared with reference standards.
Woolfolk et al (1962) found that potassium p-phenylazophenoxide
was a suitable derivatizing agent for alkyl halides, forming the alkyl
ether. Refluxing the sample and the phenoxide in N,N-dimethylformamide
for one hour and work up gave the following solid derivatives with
m.p. in °C:
Methyl 52 Hexyl 58
Ethyl 72 Heptyl 69
Propyl 60 Octyl 73
Allyl 51 Decyl 64
Butyl 61 Hexadecyl 80
i-Butyl 63 Octadecyl 84
3-Methyl-butyl 37
Ethylene dibromide gave a mixture of mono- and di- derivatives, mp
196-8°. Cyclohexyl bromide and tertiary halides did not react.
Takacs et al (1962) analyzed a mixture of methyl and ethyl bromides
by gas liquid chromatography. Relative retention times were 1.0 for
288
-------
methyl and 1.58 for ethyl under these conditions: column temperature,
52°C; carrier gas, H2 at 75 ml/min and 1.0 kg/sq. cm. (1 atm);
column, 5-mm i.d. by 3-m long; column packing, 20% B,B'-hydroxydipro-
pionitrile on 30-60 mesh firebrick; detector, thermal conductivity cell
at 52°C.
Castro (1964) gave a detailed description for the determination
of methyl bromide in organic material which involved alkali/peroxide
degradation and iodometric titration.
Bielorai and Alumot (1965) determined ethylene dibromide in or-
ganic materials by distilling 5-10 ml of benzene from a 2-1 flask con-
taining 100-300 g of sample and one liter of water, followed by gas
liquid chromatography of the benzene. Results compared well with de-
composition/titrimetry but were not as precise.
Berck (1965) used a 1/4-in. o.d. by 6-foot stainless steel column
packed with 10% SE-30 on Diatoport S (60-80 mesh) to study conditions
for separating methyl bromide, bromoform, ethyl bromide, ethylene
dibromide, l-,2-, and 1,3-dibromopropane, 1- and 2-bromobutane, 1-
and 2-bromopentane.
Perry (1966) worked out the conditions for the use of an electron
capture detector in the analysis of ethylene dibromide in gasoline.
He used a 1/4-in. i.d. by 10-foot column packed with 5% Apiezon "L"
and 0.5% polyethylene glycol 4000 on "Embacel" (100-120 mesh). The
column and detector were maintained at 95°C. The carrier gas was
nitrogen at 100 ml/min through the column, but only 15 ml/min through
the detector to avoid overloading it. Elution time was six minutes.
Reproducibility at the 30 ppm level was ± 3 ppm. The useful range
was 1-50 ppm.
289
-------
Chaudri and Hudson (1967) reported the following relative retention
times for the separation of the isomeric butyl bromides on a glc
column: t-butyl - 1.00, s-butyl - 1.94, i-butyl - 2.07, and n-butyl -
2.91. They used 1/16-in. o.d. by 4-m column packed with squalane (J0%)
on Chromosorb W, with nitrogen carrier gas at 15 ml/min and operating
temperature of 20°C.
Getzendaner et al (1968) used a commercial X-ray fluorescence in-
strument to determine total bromide content of dry organic materials
such as cereals and beans treated with methyl bromide. Calibration
curves were obtained by analyzing material previously analyzed by
chemical methods. At the 34 ppm level, precision ~vas ± 10%.
Viel et al (1969) analyzed atmospheric methyl bromide by passing
20-40 1 of air through twin absorbers (in ice) containing 20 ml each
of freshly distilled diethylamine, then combining the contents of each
bottle and evaporating the amine on a water bath. The residual hydro-
gen bromide was then dissolved in 5 ml of a buffer consisting of 1
part of IN sodium hydrpxide and 1.3 parts of IN acetic acid (V/V).
Then was added 1 ml of a solution consisting of 1 part of the buffer
and 1/20 part of a p'henolsulfophthalein solution (V/V) . Then 1 ml of
a 0.005 N aqueous chloramine T solution was rapidly added and let sit
30 seconds; 2 drops of 25% aqueous sodium thiosulfate were added to
stop the reaction. The volume was adjusted to 10 ml and the optical
density measured at 570 nm in a 1-cm cell. Amount of bromide was
read from a curve prepared from 4-10 yg of bromide. The sensitivity
limit was 4 yg, and the results were reproducible providing the HBr
contacted no organic residues. Air contamination levels of 10-20 yg/1
gave 90% recoveries, but 1-2 yg/1 gave lower, and variable, results.
290
-------
Malone (1970) described in detail an acid reflux procedure fur
extracting methyl bromide and ethylene dibromide from fumigated grain.
Preliminary grinding was unnecessary for the EDB and detriment,i! Mr
MB. The apparatus consisted of a one-1 flask with N2 inlet, a condenser
with circulating 60°C (no higher) water, Teflon tube connection to a
column of Chromosorb W (to remove traces of water), and a volunietr i i
containing isooctane immersed in dry ice-acetone. The procedure w-as to
add 100 g of sample to the swirled flask containing 530 ml v,».ater, 60
ml of IN sulfuric acid, 10 ml of 20% phosphotungstic acid, and 1/2 ml
of DC Antifoam FG-10 (spray antifoam was found unacceptable in con-
nection with the subsequent glc). With a 25-30 ml/min flow of N?,
boil gently for two hours. Popping off of the tubing connection on
top of the condenser meant that too much water had carried over, ex-
ceeded the capacity of the drying agent, and blocked the gas flow by
freezing in the receiver. Rinse four 1-ml portions of isooctane
through the tubing-dryiag agent using the N2 pressure. Allow the
isooctane solution to come to ambient, make up to volume, and inject
a 5 yl sample into a 6-ft. x 4-mm i.d. column packed with 30% DC-200
on 80-100 mesh Gas Chrom Q. Operate the injection port at 150°C, the
column at 70°C, and the electron capture detector at 200°C. Use a
60 ml/min flow of N2 carrier. Unresolved problems of other volatile
components in the grain held sensitivity to 3 ppm of methyl bromide
and 0.3 ppm of ethylene dibromide.
Muthu et al (1971) used a bio-assay method for field determination
of atmospheric methyl bromide (ethylene dibromide was tested and
found unsuitable). They placed 30 adult red flour beetles, Tribolium
-------
castaneum, in a U-tube and pumped the air sample through until all the
beetles were "knocked-down", and noted the time required. Concentra-
tion was determined by dividing this time (in hours) into the pre-
determined C.T. product. The latter is found by averaging the values
of knock-down time in hours multiplied by concentration in mg/i, using
a range of concentrations. Their range for computing C.T. was 9-58 mg/1
for methyl bromide. They tested chemically analyzed concentrations of
about 2, 5, 10, and 52 mg/1 and found quite acceptable values. Ethylene
dibromide was unsuitable because it does not produce immediate kill.
Reilly (1971) showed in some preliminary modifications to a
commercial methyl bromide leak detector that it was possible to make
it useful at the TLV of 10 ppm by using propylene instead of propane
as fuel and a ventilated copper tube reducer instead of the copper plate
supplied.
Freedman et al (1973) demonstrated that standard charcoal filter
respirator cartridges had a useful life of only one minute for methyl
bromide and 17 minutes for ethyl bromide at a concentration of 50 and
5 times, respectively, the TLV's.
VII. CHEMICAL REACTIVITY
A. Environmental and use associated reactions
Ethylene dibromide's major use as a lead scavenger in leaded
gasoline is to provide bromide atoms for the lead deposits, the lead
bromide being volatile at engine operating temperatures. Presumably
the bromohydrocarbon is decomposed at these temperatures, as Kirk-
Othmer states that decomposition to vinyl bromide and HBr occurs at
340-370°C.
-------
The chemical intermediate uses of the various bromohydrocarbons
(RBr) rely upon their relatively weak carbon-bromine bonds. The
bromine atom is readily displaced by 0, N, S, and carbanions, these
being said to be "alkylated". The RBr also readily react with finely
divided Mg or Li to form RMgBr or RLi and LiBr; these so-called
Grignard reagents are then used to attach the R group to carbon atoms
which are far more electrically "positive" than the carbanions men-
tioned above.
Levine et al (1964) compared the ability of the various isomers
of bromobutane to react with nitrogen dioxide and sunlight to form
ozone. Table 3 is an adaptation of their table of results. They did
not comment on the chemical fate of the bromobutanes. Butane itself
Table 3 — Oione Generated by Sun-
light Irradiation of 200 Pphm Butyl
Halide + 100 Pphm NO2
Induction
Butyl Period
Compound (Min)
N-butanc (control)
N-butane (control)
N-butyl for Jinide
N-bulyl lir irniiU'
\-but,yl for ii
Isobut\l li o
iHollllH 1 I> ni
See-but U br
Sec-hut \ 1 l)i
Si'f-butyl Im
i.lc
lido
nth-
iide
miiie
iiiidc
nido
lYrl-lmtyl bnnniilc
XcH~but)l bromide
Tort-butyl hrninule
65
00
35
25
35
25
;«)
no
•M
45
2.J
IK)
40
60
Miixiinum
Oi(pphm)
76
74
27
28
20
3-1
32
37
23
17
28
12
15
16
Reprinted with permission from J. A^r
MBii^Contr._Asso£._ 14:220-237C6pynnht
by Air Pollution Control Association
generated much more ozone than any of its monobromo derivatives. The
amount of ozone was corrected for normal ozone decay in a corresponding
time period.
B. Aspects with biological implications
Clegg and Lewis (1953) treated barley, beans, groundnuts, maize,
peas, rice, milled wheat, and whole wheat with methyl bromide, and did
293
-------
not tind any losses in nicotinic acid, riboflavin, or thiamine content.
From in vitro treatment of solutions of nicotinic acid, nicotinamide,
or thiamine with methyl bromide, they found apparent N-methylation of
nicotinamide, < 4%, and free bromide in the solutions of the others.
Eaks and Sinclair (1955) found that ethylene dibromide acted as
a ripener for avocados which had been fumigated with it.
Winteringham et al (1955) exposed samples of whole wheat flour
of 12.5% moisture content (one batch of which had been prepared from
wheat grown using S-35), and wheat gluten of 5 or 13% moisture con-
tent, to methyl bromide (C-14) for about 40 hours at 20°C. The samples
were then aerated to remove free methyl bromide.
After a C-14 determination on a whole sample of flour, another
sample was separated into fat, starch, gluten, and aqueous washes
for individual C-14 measurements. The gluten samples were analyzed
for N-, 0-, and S-methylation. The results are in Table 4. The
gluten (protein) fraction contained most of the C-14, the fat the
least. Higher moisture content in the gluten itself decreased incor-
poration of methyl bromide. The C-14 activity in the aqueous washes
was attributed to methanol via hydrolysis, and dimethylsulfide from
thermal cracking of dimethylsulfonium salts (only the volatile compo-
nents of the aqueous washes were determined). No S-35 was associated
with N-methylated amino acids, A separate experiment involving fumiga-
tion of the S-35 flour indicated that dimethylsulfide was evolved
naturally at ambient temperature, especially at higher humidity.
It was later shown (pp 261-8) that the principal (75%) reaction
was with histidine; 1-N-methyl-, 3-N-methyl-, and 1,3-N-dimethylhistidines
-------
Disinflation of combmed
Staple
Table <+.
'C in wheat fiacttoHs following exposure to uCHsUr
C, Wheat gluten
A, Milled whole
wbcat
li, Hiitnl whole
wbeat Kr«\vn °*'
Moisture content, °i
Friction anil meHwd ol "C recovery
(i) Whole wheat; total 14C by wet
oxidation
(2) Fat ; toUl '«(.' by wot oxidation
(3) Atjiicoui washing , volatile 1JC by
distillation through combustion
train (iree "t ll/OH)
(4) Staich , total "I l>> wot oxidation
(5) Gluten , total >*C by wet oxidation
(52) Gluten; —-O-14CH,. by difference,
(5« — 5* — 5')
(5(1) Gluten , —b-'VII.,; by decuiliposi-
tion with N.iOll followed by 11CI
*
(5«) Gluten; —S('f*C'llJ)( II, : by decotw-
j.oMtion «it(> Nadll
(5)
(5«) Gluten; —O-'4CHa + S-'TH, +
S-C'CiyCIl, , liy HI hvdn.lysih
(5/J CUiti-ii ; —X-'HUj, by dfn>iiijto:,i-
tion in 111 i- Ml ,1 (1-iieiliKli)
(54") t'.hucn ; - N-'Hll,, dy wet oxul.i-
tiou of 111 h\di>ilvs.ite ii-.ed lur (51')
«!"3 . '2 i ..''".
p.p m. of As "0 m, oi
true nun
520
164
16
1087
171
107
555
*54
0-6
3-0
1O-I
8(1-3
loo-o
As »i, of
tOtill "C
of gluten
1O-I
0-4
3*9
50-6
loo-o
Spoilt
3»
fa
153
833
374
IH'4
lOO'O
AS ";, of
tot.il "C
of yluteu
64-2
J21O IOO-O
As % of
total "C
recovered
in all
1351 loo-o
As % ol
29. 13-2
53 2-4
548 24-8
J3»f> __5
894 40-4
781
1046
ipi
I 12
36-!
77"
575
241)
5 BO
As % of
tulal "C
of gluten
7'5
»-3
26-8
loo-o
42. 6
were Isolated. It was concluded that loss of the semi-essential amino
acid histidine by N-methylation was negligible from normal fumigation.
After consideration of literature relevant to consumption of
methylated histidines and conjecture on human metabolic products (pp
269-73), It was decided that it was unlikely that methyl bromide fumiga-
tion of wheat would have toxic effects .
Siesto (1956) tested the effect of methyl bromide fumigation on
the thiamine and riboflavin content of almond, nut, and pine seed
meals. None of the treatments affected the riboflavin. Fumigation at
-------
the 5 mg/1 level at 18°C for seven days followed by seven days aera-
tion had no effect on thiamine. The 1 g/1 level for three days and
seven days aeration reduced the thiamine to 50, 47, and 33% of
unfumigated-but-aerated levels, respectively. Fumigation at the 1 g/1
level for seven days reduced the thiamine to 25, 46, and 25% of un-
treated levels.
Bridges (1956) fumigated whole wheat for 48 hours with 29.5 mg/1
of ethylene dibromide (Br-82); milled wheat, wheat gluten, and wheat
starch were exposed at the 36 mg/1 level. After airing, portions were
heated at 180-200°C to simulate baking (one-half hour).
Pre-heating levels of water soluble bromide ware low in comparison
with methyl bromide, being concentrated in the gluten. After heating,
these levels increased. The heating converted the ethylene dibromide
adsorbed on the grain or fractions thereof into ethylene glycol and
inorganic bromide. Some ether and/or ester formation occurred.
It was concluded that proper airing was very important for wheat
fumigated with ethylene dibromide, and such being carried out, there
should be little worry about residual fumigant or ethylene glycol
baking byproduct.
Iwata and Sakurai (1956) tested methyl bromide on a variety of
materials, measuring before and after bromide content: albumin 2.45
and 4.09%, casein 0.02 and 1.25%, gluten 0.06 and 0.73%, potato starch
0.08 and 0.09%, rice starch 0.02 and 0.05%, wheat starch 0.05 and 0.04%,
defatted soybean meal 0.04 and 0.88% (the treatment of this meal de-
creased water-soluble nitrogen from 5.05 to 2.92 dry weight per cent).
After treating coconut, linseed, and soybean oiJs followed by suction
-------
evaporation of residual bromohydrocarbon, they found only a small
change in the acid, iodine, and saponification numbers, viscosity, and
bromide content.
Nachtomi (1972) recovered the microsome-supernatant fractions Jrom
the centrifugation of the homogenized livers of rats and chickens,
Ethylene dibromide had no effect on the peroxidatlon ability of the
lipids (catalyzed by NADPH) from rats, but inhibited the reaction oi
the lipids from chickens .
VIII. BIOLOGY
Winteringham and Barnes (1955) reviewed the literature on methyl
bromide and ethylene dibromide, finding little that was pertinent to
this section.
A. Metabolic Effects
1. absorption
Winteringham and Barnes (1955) in a review found that experiments
with radioactive methyl bromide indicated it entered insects through
their breathing apparatus and was also absorbed and decomposed on their
skin. In another experiment no simple correlation was found between
susceptibility to methyl bromide and respiratory activity of different
stages of the confused flour beetle. Another species of this beetle
genus was made more susceptible to methyl bromide by adding carbon
dioxide to the atmosphere, a treatment known to increase the size of
the openings of the air entrances in some insects.
It was known that skin contact of liquid methyl bromide in man
caused blistering, but apparently no inquiry into possible systemic
poisoning following such incidents had been done.
297
-------
No studies had been conducted on ethylene dibromide.
2. excretion
Thomson et al (1958) gave rats s.c. injections of 1.25 g/kg of
ethyl bromide. In the urine was found ethyl mercapturic acid,
C2Hc,SCH2CH(NH-COCH3) C02H .
Bray and James (1958) reported that rabbits dosed with bromohydro-
carbons excreted mercapturic acids in their urine. They identified
only butyl mercapturic acid, from dosing with 1-bromobutane, but did
find mercapturic acids from dosing with 2-bromobutane, bromocyclohexarie,
1-bromo-heptane, -hexane, -octane, and -pentane. The longer the alkyl
chain the lower the percentage of the dose eliminated as a irercapturic
acid.
Grenby and Young (1959) isolated n-propylmercapturic acid from
the urine of s.c. dosed guinea pigs, mice, rabbits, and rats.
Bray and James (1960) reported more results of their earlier (1958)
study and follow up. Among these were the identification of pentyl
and hexyl mercapturic acids from rabbit and rat urine. A second
metabolite from rabbit urine after 1-broinobutane dosage seemed to be
a peptide of S-buty1-L-cysteine and glycine. A third metabolite from
this source seemed to be a more complex peptiae containing S-buty1-L-
cysteine, glutamic acid, glycine, and an unidentified sulfur compound.
This third metabolite was the only one found in guinea pig urine after
dosage with 1-bromobutane.
Grenby and Young (1960) published complete details of their 1959
report. They added that there was no evidence for the propyl mercap-
turic acid having been formed during the acid treatment of the urine
-------
as a hydrolysis product of a mercapturic acid precursor, such as is
seen with aromatic mercapturic acids.
They found that, in vitro, n-propyImercapturic acid was dearetylatud
by extracts of rat kidney or liver, but did not care to state posi-
tively that this may have occurred in vivo. Only 1/4-1% of the ad-
ministered dose was isolated as pure mercapturic acid, but it was not
thought that enough was lost in handling and purification to deny that
this method of metabolism of the bromohydrocarbon is subordinate to
still undiscovered pathways.
Thomson and Young (1960) reported finding in the urine of rats
dosed with bromoethane (in addition to ethyl mercapturic acid) ethyl
mercapturic acid-S-oxide, C2H5SOCH2CH(NHCOCH3)C02H.
Janes (1961) in still further elaboration of her 1958 study with
Bray found that only negligible quantities of heptyl, and none at all
of octyl mercapturic acid are present "as such" in rabbit urine after
dosing with the appropriate bromohydrocarbon. Something is present
which reacts the same with the nitroprusside detecting agent as free
alkyl mercapturic acids do.
Bray et al (1964) dosed rabbits and rats with butyl, pentyl,
hexyl, and heptyl bromides. They isolated from combined urines these
purified mercapturic acids: butyl - 2% of dose in rabbits, 4% in
rats; pentyl - 0.5% of dose in rabbits, 1.7% in rats; hexyl - 0.2% of
dose in rats; heptyl - could not be crystallized (from rats), and
could not be detected from rabbits (except after acid treatment and
ouly in small amounts).
Octyl mercapturic acid could not be detected, dJrectJy or indi rec L 1 v ,
-------
after dosage to either rabbits or rats. The same was found for butyl
and hexyl mercapturic acids after dosage to guinea pigs. Table 5
contains their results including urinary bromide recovery from dosing
with sodium bromide or bromoalkane.
Barnsley et al (1964) reported the metabolic pathways for ethyl
bromide in rats diagrammed in Figure 1.
NH-CO'C'Hj NH,
CIVCKj-SOCHj-CH - - — CH3-Cir.,'SO-CHt-CII
(l)* iozH urt i. Biosynthesis of ethylmercapturic acid sulphoxide. Conversions demonstrated in the animal body
are shown by solid arrows and possible conversions are indicated by broken arrows,
Barnsley (1964) reported finding in the urine of rats dosed with
1-bromopropane (in addition to 1-propyl mercapturic acid and 1-propyl
mercapturic acid-S-oxide) 2-hydroxypropyl mercapturic acid. The yield
was only 80 mg of the dieyclohexylammonium salt, from the combined
urine of 32 rats given a total of 54 g bromopropanes .
Nachtomi et al (1966) gave rats stomach tube doses of ethylene
dibromide, 100 mg/kg, as 2% solutions in soybean oil. The main urinary
metabolite was 8-hydroxyethyl mercapturic acid; in much smaller amount
was found S-(6-hydroxyethyl)-L-cysteine .
Barnsley et al (1966) were unable to detect urinary sulfur-
containing metabolites of 2-bromopropane given to rats by s.c. injec-
tion, except as traces and not consistently.
-------
Table 5. Excretion of mercapturic acid and bromide by animals given bromoalkanes, or sodium bromide
Compound
1-Bromopropane
1-Bromobutane
1-Bromopentane
1-B romohexane
1-Bromoheptane
1-Brorno-oetane
Sodium bromide
Rabbit
Dose Mercapt- Apparent Bromide Bromide
(m-moles/ uric mercapt- after
kg.) acid uric acid (96 hr,) 4 days
1-9 2-7 -
(1-3-5-2)9
1-7 4-7 41 16 43
(3-9-5-7)8(27-54)20 (11-21)6 (41, 44)2
1-5 2'3 21 27 50
(1-6-3-7)9 (16-23) 3 (18-40)3 (49-52) 3
1-3 0-9 23 29 48
(0-3-2-3)7(21-25)3 (17-42) ** (45-S3)3
1-1 N.D.6 16 20 66
(12-19)3 (11-22)4 (63-71)3
1-1 N.D.6 12 19 53
(0-21)5 (17-20)3 (47, 58)2
1-7 - - 12 48
(22, 25f (44-55)3
Rat
Dose Mercapt- Apparent
(m-moles/ uric mercapt-
acid uric acid
kg.)
2-3
2-3
2-1
2-0
1-9
1-7
5*1
(4-7-5-7)6
6-3 54
(4'0-8'l)8(21-72)16
5-9 35
(2-8-9-0)7 (14-55)6
3-1 52
(1-6-5-4)u(23-69)6
2-3 70
(1-0-3-if (40-90)6
N.D.*
46
Guinea Pig
Dose Bromide
(m-moles/
kg.) (24 hr.)
1-2
1-0
1-0
0-9
0«7
2-5
Amounts excreted are expressed as percentages of the dose, ranges are given in parentheses and numbers of
experiments are indicated by superscript numbers. Unless the times are given, the results are for the amount
excreted until the 24 hr. excretion returned to the normal level. N.D. indicates not detected; —indicates not
examined. In the guinea pig, the excretion of true mercapturic acid was examined after administration of all
the alkanes listed but none was detected. The value for apparent mercapturic acid was 18 (10-12)-" from bromc-
vuta~e (dose I'D m-mole/kg.) . _ - , , . ,
Reprinted with permission from Blochem.
iL. 90:127-32 (1964). Copyright by the
Bioch-rnical Society.
13
(9-16)'
18
do, 19 r
-------
James et al (1967) dosed rabbits with bromocyciopentane (A), -hexaiie
(B), and -heptane (C). A]1 gave methyl bromocyc]oal^yl triacetylgluco-
siduronates in the urine; in the case of B this metabolite was shown
to be the 2-bromocyclohexyl isomer.
Also found in the urine from A-dosage were cyclopentyl mercapturic
acid, 2-hydroxycyclopentyl mercapturic acid (also a metabolite from
cyclopentene and epoxycyclopentane dosage), and another sulfur-containing
material (also a metabolite from cyclupentene and epoxycyclopeiitane,
and forming in vitro from cyclopentanone and N-acetyIcysteine) .
The major sulfur-containing urinary metabolite from B-dosage was
an unknown material (also a metabolite of cyclohexene) which was not
the 2-hydroxycyclohexyl mercapturic acid (a metabolite of epoxycyclo-
hexane); in traces was found cyclohexyImercapturic acid.
Also found from C-dosage were small amounts of cycloheptyl mer-
capturic acid (a metabolite of cycloheptene), traces of 2-hydroxycyclo-
heptyl mercapturic acid (also a metabolite of cycloheptene and epoxy-
cycloheptane), and another sulfur-containing compound (also d metabolite
of cycloheptene and epoxycycloheptane).
James et al (1968) gave female rabbits and rats stomach tube doses
of aqueous suspensions of 1-bromobutarie . The metabolites found in
the urine are indicated in Figure 2. The rabbits excreted 8% of the
dose as butyl mercapturic acid and 14% as hydroxybutyl mercapturic acids;
the corresponding figures for rats were 7 and 22.
In a separate experiment bile duct-cannulated rats were given s.c.
doses of 1-bromobutane. In the bile were found the metabolites S-
butylglutathione, S-butyl-cysteine, and S-butylcysteinylglycIne. Urine
-------
collected over the same time period contained butyl and hydroxybutyl
mercapturic acids,
A supernatant of rabbit liver homogenate produced S-butylglurathione
and traces of S-butylcysteine when mixed with 1-bromobutane. Rabbit and
rat liver slices produced 3-hydroxybutyl mercapturic acid from butyl
mercapturic acid and from S-butylcysteine. Only rat liver slices pro-
duced 2-hydroxybutyl mercapturic acid. Rabbit liver slices produced 3-
(butylthio)lactic acid, VIII in Figure 2, from S-butylcysteine.
XH-
I "
KH.CO-Cir,.Cll..CH.t'O,H
I
C'Hj.cir-.rir..ni...s.cH..rit
',
m
Kir.
^Hn- OH
CH3.Clt..C'H2'Ciri.S.CH;.CH '—> CH3-CH~.Cir,.CH».S.CH-CU
COjU COM
CH3.CH,.eH.CH. <1U> (VIII)
" V "
(IX)
WCHj.CHj.CH«.CH-.S-CH->-CH
4- " 1
WiH O COgH
(IV) (VII)
f
'jib
XH-CO-CIIj
C'H3-CII2.C!r.CfIu..S.CIT2.CJ[ CH3-C!I-CH-..CH.-.S.CHo.CH
I i t " ' I
OH C02II OK CQjH
(V) (VI)
Fi'guce 3. JlctJioIi^m of l-hroinolnitatie. All tho i;«otions Lavo been shown to occur in the rat,
am! ttio*o marUftl Hb in tlio tabbit.
Reprinted with permission from Biochem
.h. 109:727-36 (1968). CopyrighrbYlh?
Biochemical Society.
-------
Jones and Edwards (1968) gave rats oral doses of ethylene dibromide
with both carbon atoms being C-14. They isolated 20% of the dose as
C02. Metabolites found in the urine were S-(Z-hydroxyethyl)cysteine,
the S-oxide of the latter, hydroxyethyl mercapturic acid, and the S-
oxide of the latter.
Jones and Howe (1968) added long-chain bromohydrocarbons to the.
yeast Torulopsis gropengiesseri in a glucose-containing medium. Methan-
olysis of the resultant glycolipids gave a,w- alkane dioic acids
(dimethyl esters). Starting bromohydrocarbon, product(s), and yield(s)
were as follows: 013(012)14, (CH2)±3(C02-)2, trace
CH3(CH2)15, (CH2)11+(C02-)2, 50%
CH3(CH2)16, (CH2)15(C02-)2, 45%
GH3(CH2)17, (CH2)16(C02-)2, 17%
& (CH2) 11+(C02-)2, 29% (byproduct)
CH3(CH2)17, (CH2)16(C02-)2, 26%a
& (CH2)11+(C02-)2, 9%b
CH3(CH2)19, (CH2)11+(C02-)2, 34%
CH3(CH2)21, (CH2)lit(C02-)2, 36%
a - starting material and product both A^ olefins
b - A7 olefin byproduct of A9 starting material
From CH3(CH2) 13CH(CH3)Br was obtained a low yield of H02C(CH2) j 3OI(CH3)Br,
but no glycolipids. From CH3(CH2)13C(CH3)CH2Br was obtained a 50% yield
of H02C(CH2)13C(CH3)C02H (as the dimethyl ester).
Nachtomi (1970) gave rats stomach tube doses of ethylerte dibromide.
From the supernatant of the homogenized liver were isolated S,S'-
ethylene bisglutathione, S-(2-hydroxyethyl)glutathione -S-oxide, and
30h
-------
S-(2-hydroxyethyl)glutathlone; from the similarly treated kidneys were
isolated S-(2-hydroxyethyl)glutathione and 2-hydroxyethyl mercapturic
acid. When the bromohydrocarbon contained C-14 and the rats were
sacrificed four hours after dosage, 6% of the C-14 war. recovered (63%
in the liver, 37% in the kidneys).
Kaye and Young (1970) injected rats with allyl bromide and found
allyl mercapturic acid in the urine.
James and Needham (19.70) found 4-carboxybutyl mercapturic acid
in the urine of rabbits dosed with bromopentane and in the urine of
rats dosed with 1-bromopentane or 1,5-dibromopentane.
James et al (1970) administered aqueous suspensions of bromocyelo-
alkanes via stomach tube to female rabbits and examined their urine
for metabolites. Sulfur-bearing metabolites are discussed in James
et al (1971) .
Bromocyclopentane gave bromoeyclopentyl-tri~0-acetyl-D-
glucosiduronic acid (isolated as the methyl ester). Bromocyclohexane
and -heptane gave analogous compounds. Table 6 incorporates the re-
sults of urine analysis for total bromide, glucosiduronir acid, and
ethereal sulfate.
TABLL i,. AMOUM ot MI-TABOLITLS LXCREIUJ i.\ URIM: BY RABBITS
Compound administered
Bromocyclopentane
B ro mocy clo hexa ne
Bromocycloheptane
\
Dose
(m-rnote/kg)
1-8
1-6
1-5
Total'
hjomide
62
(54-73),,
6*
(5S-76)«
6-i
(56^7 Ijj
GlucosKluionic
acid
27
(16-38)0
61
(35-84)5
78
(69-85)6
Ethereal
sulphate
13
(12-16).
9
(8-10)3
19
(17-22)3
mission from Biochem.
Pharmacol. 19:743-49'
(1970). Copyrinht by
(8-10)3 Pergammon Press Ltd".
Results are mean^ expressed n-, pciccniaae of JL">C, uiih langes in parentheses; the numbers of
enninatic-ns arc ii)i1ic,:ted b;, inferior fi«ure«
Tins includes the bic.nnd,- uvcicicil pkn orj.-ivoIU hound bioniinj v.hic'1 1-5 .iNo cstiuiatcd a>
bromide.
As measured by percentage of dose excreted as bromine, after 72
-------
hours the order of metabolism was: cycloheptane = cyclohexane >
cyclopentane > 1-pentane = 1-hexane > sodium bromide (all at same molar
dose of Br). After 96 hours the only change was the near equivalence
of the cyclopentane to the first two (the range for all compounds at
this time being 47-64%).
Degradation of the glucosiduronic acid metabolites indicated that
the rings had been attacked by oxygen at the carbon atom adjacent to
that bearing the bromine atom.
James et al (1971) dosed rabbits and rats with bromocycloalkanes
and examined the urine for metabolites . For the non-sulfur-bearing
metabolites of the rabbits see James et al (1970). Tneir findings on
mercapturic acid metabolites are in Table 7.
1 A11U 7. '^AC't' I1ON 01 VI HCAITU'UC AC!P AN'n IIVIM-OX1! N" 'TA'TUIUC ACIDT BY DOSrD RAlltlllS ANI> RAT1
K '.Mul Km
>-• Al*\l r. *-."-()»' .v/.'n-MJH 1-UH-AlLyl Alky! n'i-2-Oll
A'kvl Al >1 AlKyl
r/-^'M 2-01! J-1
Al, >1
:-s <"; ft: 21 2 i o is r 7 '?•;
(1-6-20)' (0-6-U9)4 (00-C9)* (1D-0-:?. 2)» (O-S-1 2)* (1-1-22)* (l-J-il)J ( : 6 : ''.
!C j 5 111
ir tr Ml (99-llj1 NO NO ND (!0.-:2
12' 5506 ••. :
(1-2-1-3)5 ND vn (5 5)3 (0-1 0J MD ND (3 V :•)'
Reprinted with permission from Biochem. Pharmacol. 20:897-907 (1971). Copyright
by Pergamon Press Ltd.
§- Includes trace amounts of other hydroxymercapturic acids.
Amounts are expressed as percentages of the dose, ranges are
given in parentheses and numbers of experiments are indicated
by superscript numbers. ND indicates not detected; tr indicates
trace amounts.
No S-oxides were detected. No other S-containing metabolites
were detected by running the experiment on rats fed yeast labelled
with S-35.
Grasse and James (1972) dosed rats with 1-bromopentane and
306
-------
determined the metabolites with the assistance of [I-14C]-1-bromopentane.
Stomach tube aqueous suspension doses were given to adult female rats
(only one rat received the radio-bromopentane). No S-oxidew won-
found. Table 8 contains their findings of a positive nature, Metabo-
lite 2 consisted of 2-, 3-, and 4-hydroxypentyl mercapturic acids. It
was hypothesized that Metabolite 4 was a hydroxy derivative of 4-
carboxybutyl mercapturic acid. The feces of the 24-hour period follow-
ing dosage contained only 0,4% of the radioactivity present in the
radio-bromopentane dose. No metabolites were detected in 72-hour ieret>
after unlabelled bromopentane dosage.
Gas chrotnatography of the hydroxy metabolites as methyl esters
gave the following relative peak areas: 4-hydroxy, 1; 3-hydroxy, 3;
2-hydroxy, 4; sulfate ester of the 3-hydroxy, 16; sulfate ester oi the
2~hydroxy» 21. Until the detector sensitivity of these compounds has
been determined, these figures should only be read as implying that
the 2- and 3-hydroxypentyl mercapturic acids are excreted mostly as
sulfate esters.
Table 8. Metabolites of 1-bromopentane excreted by the dosed rat.
(a) (6)
1-Bromopentane ll-J'C]-l-J(L-onH>prnt,!rie
(% dose) {% (!UM: of actn.it,>)*
'Material drtern:in< d
Pentylmcrcaptunc acul 7-0 (4-9-10-6),, 4-5
Metabolite 2 (hydroMpentylmereapturic acid) 8-9 (6-8-12 0),j S-2
4-Carbo.\ybutylmca.aptunc acid 2-4 (2-2-3-0)k 2-4
Metabolite 4 ii.d.', 1-1
1-Bromopentanet 1-4 (1-3, 1-4), u.d,
CO.t .'' n.d. " 62-4
Bromide § 38 (3? 42), n.d.
Results are means expressed as pert enlaces of tlixc \vith rnn»cs in parenthesis and
numbers uf ammuL a% subscripts,
* Activity of dttoc \v;i» 1-18 x 10" il.p.m.
f Excreted in expired air in 5 h after dose.
\ 52% in 0-24 It and 10 4°;, in 24-120 h after dose.
§ Excreted 0-72 h after the dose when excretion was still incomplete. Rats dosed
with an equivalent amount of XaBr excreted 31",, (28-3 J)j of the bromide in 72 h.
^n.d.=,notd,-,,.rmm,.,i. Reprinted with permission from Xeno
bi.Q.t|ca 2:117-27 (1972). Co)>yriY,Kt
Taylor X I"nine is 1 td.
-------
Sulfur-containing metabolites accounted for only 74% of those
present in the urine after 24 hours - determined from the radio-
bromopentane experiment.
3. transport
Getzendaner (1965) gave chickens a layer ration containing an
eight-fold range of bromide residue from methyl bromide fumigation.
The bromide content of the whole eggs produced reached the same level,
roughly, as that in the feed in 20-36 days. Figure 3 depicts the data
graphically.
24 32 -10 4U
DAYS ON FUMIGATfD FEED
Figure
3. Bromide residues in egys from chickens on
gated diets
\ Control
X 51 -p.p m. diet
D 102-p.p.m. diol
A 161 'p.p.m. diet
6 206-p.p.m. diol
O 410-p.p.m. diet
Reprinted with permission from J. Agri.
Food Chem.. 13:349-52 (1965). Copyright
by the American Chemical Society.
-------
4. distribution
Getzendaner (1965) gave chickens a layer ration containing an
eight-fold range of bromide residue from methyl bromide fumigation.
His results on distribution of bromide in various portions of the
hens and their eggs are presented in. Figures 4 and 5, Tables 9, LO,
11, and 12. In 1965, the author commented, it was common practice
to incorporate hydrolyzed chicken feathers as up to 2% of cattle feea
and 5% in chicken feed. Since the average feather analysis for
bromide residue was lower than that in the feed, there was no cause for
concern about bioaccumulation.
sro
150 200 250 3OO 330 40O
PPM IN FEED
100 150 200 250 30O iso
PPM IN FEEO
Fi'gure 4. Residues of bromide in eggs vs. bromide content Figure 5. Residues of bromide in chicken f <•
of feed days on fumigated diets
400 -ilO
o'v- 70
n Wholo <
O Egg shei
3 Yolks
X Whitei
O Ligh! meat
X Dark meat
• Liver
A Skin
H FL'.jtIiorj
A KifJnoy
-------
Table 7. Dromidc Residues Found Table 10. Rcte of Bromide Accu-
in Egg White and Yolk mulation offer 70 Days on Feed
flr Found, P.P.M.
si P:P.M 410 P P.M
Days on
Fumigo/fcd
Feed
32
33
34
35
36
in feed
In
white
32
28
26
26
24
34
In
folk
57
62
71
77
56
76
in feed »
In
white
324
280
293
290
428
276
In
yelk
506
480
45.0
441
55.0
392
Tissue
WlloK- i>x.-
(llO sliclls)
Yolks
Whiles
r-KO, sin-Ms
l-if'lu iiu ,it
Dark nil'. u
Skin
f ,i\ IT
ICidncv
lll..,,d
Ratio:
AY. P.P.M.
in Tissue
P P.M. in Feed
1 .0
' .2
0.8
0 3
0.2
0 3
0 4
O.S
Of
. (>
0.8
1 .7
ttotio:
Mar.. P.P.M.
in Tissue
P.P.M. ,n Feed
1.3
1 .5
1 1
0 5
0.23
0.5
0 5
0.7
1C
. D
o.y
2.1
Av. 30 67 315 473
Table 11. Bromide Residues in Egg
Shells
, Ooyj on Bromide in
Bromide in Fumigated fgg Shof/s,
Feed, P.P.M. Feed P.P.M.
Control ... < 1 3
Av. <13
51 46-66 <13
36
17
Av. 19
102 41-54 13
27
21
Av. 17
161 42-54 23
25
39
29
29
Av. 29
206 42-56 44
33
42
34
Av. 38
410 42-70 103
116
96
85
Av. 100
-------
T0b!e 12. Bromide Residues Found in ChicLcti TK'.uc',,
Brc.fmi:fe KiMictae*, f ^ M., founj af-f^:
Days
Treatment
and Ti%$t/0
Control
Light mi at
Daik HK.K
Li\ei
Kidneys
Skin
Feathers
Blood
51 p.p.m. lir
Li^ht meal
Park meal
Liver
Kidneys
SLin
Feathers
Blood
102 p.p.tn. Kr
Light meat
Dark meat
Liv er
Kidneys
Skin
Feathers
Blood
161 p p m. Br
Light meat
Dark meat
Liver
Kidneys
Skin
Feathers
Blood
206 p.p.m. Br
Lij;lit meat
Dark meat
Liscr
Kidneys
Skin
F'cathers
Blood
410 p.p.m. Br
Light meat
Park meat
LKcr
Kidneys
Skin
Fe.ulier*
Wood
1 5, Tables
ision from
28
1
<5
<5
6
8
<5
6
11
13
33
45
30
23
16
28
40
65
50
10
31
41
80
116
56
15
<5
8
18
26
19
22
66
100
172
344
290
34
44
Birrf
2
<5
<"5
20
<5
14
13
?6
44
18
70
96
28
42
39
66
4t
117
162
31
58
73
130
46
158
241
44
71
112
152
53
33
331
H
'^;?
<1
<5
<;s
-------
B. Physiological Effects
Winteringham and Barnes (1955) reviewed the literature on methyl
bromide and ethylene dibromide. The former produces nervous system
damage of a nonpermanent nature, and interferes with enzyme function
by reacting with SH-groups. The dibromide causes a non-narcotic type
of unconsciousness, but no apparent nervous system danage. Minor
hepatic and renal necrosis occurs.
Olomucki (1957) demonstrated that, in chickens, it was likely
that ethylene dibromide caused the pituitary gland to decrease its
production and/or release of follicle-stimulating hormone. This re-
sulted in smaller than normal follicles, smaller than normal eggs,
and, ultimately, cessation of egg production. An in vitro study
demonstrated that the dibromide had no demonstrable effect on follicle-
stimulating hormone.
Amir and Volcani (1965) administered 2 mg/kg/d of ethylene di-
bromide orally to male calves from tour days after birth to 12 months
of age, then changed the dosage to 4 mg/kg/every other day. They
collected semen samples at 14-16 months of age, thereafter once a week
for 8-10 months.
Health and growth were unaffected. Sperm density and motility
were low. Malformations of the sperm included coiled tails, no tails,
and degenerated pyriform heads. Recovery after discontinuation of
dosage required 10-90 days. Onset of appearance of malformed sperm
occurred two weeks either after first dosage to a 16-month old animal,
or after resumed dosage in a previously dosed-from-birth animal after
cessation of treatment.
-------
Johnson (1965) gave an adult female rat a stomach tube dose of
1.16 ramole/kg of ethyl bromide. After two hours the hepatic gluta-
thione level was 52% of normal. This compared with a literature value
of 50% for a 1,6 mnole/kg dose of 1-butyl bromide.
Alumot (ne'e Olomucki) and Mandel (1969) conducted additional ex-
periments with chickens regarding ethylene dibromide, egg laying and
size, and gonadotropic hormones. They decided that the conclusions
drawn earlier (Olomucki, 1957) were incorrect, and that the dibromide's
effect on chickens' egg laying was still unexplained,
Kazakova and Lis (1971) exposed mice four hours a day, five days
a week for four months to an atmosphere containing 90 yg/1 of 2-
bromopentane. They observed an inhibition of neutrophilic phagocyto-
sis, a suppressed development of local infectious inflammations, and
an increased resistance to staphyloccal sepsis.
Alumot and Harduf (1971) fed laying hens feed containing 100 ppm
ethylene dibromide. When the egg weight had dropped by one-third, the
hens were given i.v. .injections of radio-iodide labeled chick serum
globulin (CGG) or albumin (CSA). As may be seen in Figure 6 the yolks
of eggs laid after these injections incorporated only half as much of
the protein fractions as non-dibromide fed controls. Similar results
were found for incorporation of the labeled proteins into the vitelline
membrane.
In another experiment CGG or CSA was given to control and di-
bromide- fed hens, who were sacrificed after 40 hours. The control hens
had only half as many follicles as the treated hens, but the total
weight was the same and uptake of radio-iodine was double that in the
treated hens.
-------
Reprinted with permission from Cqmp_.
Blochem. Physiol. 39{lB):61-68 "097T)
Copyright by Pergamon Press Ltd,
4 6
EGG No.
Fig\WQ 6.Total "5I in yolks (Trial 1),
EDB - ethylene dibromide
James et al (1971) dosed rats with bromocycloalkanes and measured
the hepatic glutathione after 1-4 hours. As seen in Table 13 there
was a definite response to ring size.
Alumot (1972) reviewed the problems of ethylene dibromide reduction
of egg size in chickens. The conclusion was that a reduction of protein
uptake by the follicles resulted in slower growing follicles, hence,
smaller eggs,
-------
TAI.U 13.Eirccr OF SOM. MI IICAITURIC ACID r;. su-, .in' d i'l v.;itu-, ;ulmini,K-n-d at 0 lir to rats v;',,icii l.nd l.v.j f.-isr-;,-] for 19 li' i> - h
«;.-/.,..:; Coiitro! ai,:,!^!', \,f ic ,'NM'n wtid-r by iJfirr.ich lulu
IX. ENVIRONMENTAL EFFECTS
A, Persistence and/or Degradation
Coulon et al (1954) fumigated chestnuts with 17 or 118 g/cu.ra. of
methyl bromide. Analysis revealed bromide residues o£ 79-180 ppn,
highest in the albumin, and dose rather than duration of treatment re-
lated. They recommended against this fumigation.
Olomucki and Bondi (1955) measured total and non-ether soluble
bromide in samples of grain meals before and after extraction with ether.
It was shown that the fat content served as a solvent for the ethylene
dibromide used to fumigate the grain, but did not enhance the reactivity
of the fumigant with the protein content.
Viel and Giban (1958) injected different types of soil with ethylene
dibromide and left them undisturbed at or below 15°C for 8-9 weeks.
-------
There was still dibromide present after this time, in a distribution
gradient about the injection point. They recommended tillage after
fumigation and before planting.
Lindgren et al (1962) determined that the total bromide residue in
whole wheat (after fumigation with methyl bromide and aeration until no
longer effective) increased rapidly with moisture content over the range
9-15% water, especially at higher fumigant concentrations. Over this
same range higher residues were found the higher the temperature at which
fumigation had been carried out (10-32°C range studied).
They compared bromide residues in various mill fractions obtained
by milling after fumigation or fumigating after milling. In neither
instance was there a correlation with fat content of any fraction. On a
relative basis the residues in fumigated-milled fractions were: middlings -
1, flour - 1.1, whole - 1.6, shorts - 2.6, and bran - 2.9; the residues in
milled-fumigated fractions were: whole - 1, middlings -6.5, flour - 7.4,
shorts - 7.9, and bran - 8.6. The only change in the order on. going irom
post- to pre-milling was the whole grain dropping from the middle to the
bottom. However, residues in the fractions were 2-5 times higher when pre-
milled, even at slightly lower moisture content.
Sinclair et al (1962) fumigated whole and milled wheat with ethylene
dibromide. After 10 days standing at 21°C, about 97-98% of the added di-
bromide could be recovered unchanged from either type of sample. The
relative total bromine residues in milled-fumigated fractions were: whole
- 1, shorts - 2.6, middlings - 3.4, bran - 3.6, flour - 3.6+. The order
of ionized bromine residues was: whole - 1, shorts - 1.8, flour - 2.4,
bran - 2.8, and middlings - 3.3.
Sinclair e1 al (1964) showed that corn ami wheat (9% wat ?i 1 ahsnrho'l
-------
about the same amount oi e Chylene dibromide W!K 11 fumigated at 10-20ui,,
but corn absorbed more at a higher temperature. Studying ihe el feet on
fumigant retention of such variables as moist mo. content, sm^i^; 11 on tem-
perature, am! post-fund j,a t ion storage temperature, (lie auiliotj . oua 1 .ui
increased retention i rc«ui increases, iru i >asi ;, , aid do< r e...--,(_ s- , r.^|, i i i.. i
for wheat, ami i nrre.a.-.o.:, in all three tor corn.
Recovery of ethylene dibromide added to %< ound <-*orn did not Jr>
simulate current practice but slanted lo yie 1,.! ui.j \iiiial bror.iide i t-s i dues .
Some of thtM r tested goods may already have bet'n treated with Inonade
fumigants. The.fr results are in Table 14. The last, column on the rl^.ht
was intended Lo be used at; an aid in predicting losidue itccumu I at i on.s
fo7' ' epeti f i •'< i umigal ion'5. The 1966 :t,!eral r.-^istei toler.nve iev.-lb
for i-romide . i: idues in processed food, -n-erc1 p,iiiiall>.' h.isi d <>ii tie ><.•••
suit , pretM •>: I ( i! nerein.
( astru and !Je3set (l'H'j8) incubated .soi.l, nntrienis, --aid erhvleiu-
dihromide-1 ».'--i'--14. i»it!iin eight week:- nearly .ill 1^1" the lunlg.aii l;,nl
been -'onve: i c>( to etle l*n.' and bromide ions. ' -IK.-SI :ie,,o- n/ ,, 2,3
di t>r' i.iiobutaiie ...is usc.l, irans- or cis—.'--butein > vi-r-' r i odi<'-f d , 'e.-,p. , t i •/,-
Jy, indicativ., ol ster.t osrecif ic trans vlimin.iiion nf the iU '.-; . (uijnli
tative analysis of this eciapound rould not he run because ot h vdro i v;. i .s
observed in the sterile control cultures; lu'wew- r, no oLi. iLnii- or ga-.t-unt-
products were seen in the controls.
Brown and Jenkinson (1971) fumigated soil with methyl bromide at
-------
I-'ood
Candy and Contccuons
Candy but'
Brand 1
Chocolate bar
Brand 2
Chocolate bar
Brand 3
Candy bar'
Brand 4
Candy bar'
Brand 5
Fudge
Oraiid 6
Chewing yum
MarshmullowN,
large
Marshmatkms,
miniature
Minis'
l'oanut$c
Sugar, cane*
Cereal Products
Corn Hakes
Brand A
Com flakes
Brand B
Hominy grils
Oat cereal
PulFed rice
Brand A
Putted rice
Brand C
C earn of wheat
Puffed wheat
Shredded \\ Iv.-at
HaU-d cereal
Hr.ind D
Bakiiii', powder
Noodles, egg
Starch, corn
Macaroni
Dot: food, dry
Cattle Iced, ini.scJ
Cornnieal
I lour, rice
Hour, Miy
I loin, tapioca
I imii, white wheat
Bund 1' '
Table 11,
HO, •;,;
1.3
1.6
1.3
1.2
1.2
0.6
2.1
0.7
1.4
0.8
1.8
" 0.1
3.9
4.7
8.2
4.3
4.1
5.8
11.1
4 9
6 9
7.9
11.6
6.7
10.6
9 6
8.9
10.3
11.9
8.5
6 0
8.5
1 1.3
Bromide
"V.
80
SO
60
KO
m
80
80
so
80
80
SO
80
80
80
60
SO
so
80
80
80
80
SO
80
80
80
SO
80
80
80
80
80
80
80
80
80
80
80
80
SO
80
80
80
60
SO
60
SO
80
SO
80
80
60
SO
60
SO
60
SO
60
HO
XO
80
70
71)
80
SO
Ml
,Vtl
Itcsiilui's in I
1 lours
24
24
12
12
24
24
24
24
24
24
24
24
24
24
12
12
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
12
12
12
12
24
2!
24
24
12
12
12
12
12
12
12
12
24
24
42.5
42 5
24
21
: \
21
nod Commodities
Hate,
1.1../M.
1
3
3.75
2.5
1
3
1
3
1
3
1
3
3
3.75
2.5
1.5
1
3
1
3
1
1.5
I
1.5
1
1.5
1
.5
.5
.5
1.5
1
1.5
1
1.5
1
1 5
3.75
2.5
3.75
2.5
.5
1,5
3 75
2.5
3.75
2.5
3.75
2.5
3.75
2 5
.5
.5
,s
Bromide IJcUdiics, IM'.M.
1'itlrcatfd
I, 1
0,0
2, 2
1, 4
3, 2
2, 2
0, 1
0,0
0,0
1,2
4, 13
7,7
5,6
5,6
4, 3
0,0 '
0,0
0,0
12,25
0,0
0, 10
8,5
0,0
0,0
3, 3
0,0
8, 11
8,0
0, 0
106, 107
0
0
14. 16
12, 13
lriMtt.il (net)*
8, 5
26, IK
25
2ft
fi, 7
IS, 17
8, 7
28, 13
10, 8
31,31
3, 3
It, 4
4,3
9, 10
6
7
0,0
0,0
5, 1
6, 12
27,5
59,55
0,0
0,0
0,0
0,2
0. 2
0, 2
6, 11
10, 11
8,7
15, 14
7,9
6, 7
9,9
10, 14
17, 17
34, 44
6,8
9, 11
5, 8
11,4
14
7
0
0
15, 16
23, 20
3, 2
5,5
12
12
ys
81
68
81
6S
68
0,0
13,53
4l>
0
41, 41
5', 61
21, 1H
26, W
Spci-ilu
6
7 3
6 7
10
6.5
5.8
7 5
6 3
9
10
3
2 5
3.5
3.2
1 6
2.8
0
0
3
3
16
19
0
0
0
0.7
1
0 7
R 5
7 0
7.5
9.7
8
4.3
9
8
17
26
7
6 7
6.5
10
3 7
2 8
0
0
16
14
2.5
3.3
3.2
4,8
26
32
IS
32
18
27
0
1 >
4')
0
41
3H
20
19
Reprinted with permission from J. AgrjL
Food_Chem._, 16:265-71 (1968). Copyright
by the American Chemical Society.
-------
Table 14- (Continued)
I'ond
Brand 2 A'-' »
Brand 3' « ''
Brand 4-
Hour, whole \\heat
Biand 5''
Brand 6
Brand 7
Cake mix
Brand 8
Brand 9
Brand 10
Brand 11
Pancake mix
Brand 12
Brand 13
Pie crtiil mix
Btaiul 14
Brand 15
Brand 16
Annual 1'rodiu.ib, F:ats
Cheese, elieddar
Cheese, cott.ige,
creamed
Cheese, pincomimg
Cheese, parnicsan.
grated
Beef, roast, chuck
Beef, roast, loaf
rraakfurlers.
skinless
Pork, shoulder,
smoked
fork, bleak
Bacon, sliced
L:ggs, poudered
Gelatin, iniilaxoied
Ciclattiv iliuired
Milk, malted
Dry
Skimmed
M.ik, dis
SShoK-
I'oik x>uvi;-c, link
!!/), ",:
11 3
11 1
8 8
10 5
8 2
11 4
4.3
3,8
4.8
4 1
8.4
8.5
6.4
6.2
7.2
5,1
50
13
57
36
35
37
37
11.7
1.8
l'J.5
' 1'",
80
HO
80
SO
SO
SO
so
so
so
80
60
SO
60
80
80
80
80
80
SO
80
80
80
80
80
80
80
80
80
80
80
SO
SO
80
80
SO
80
80
80
80
80
80
80
80
80
80
80
SO
SO
SO
80
NO
SO
Si)
80
00
80
SO
80
W)
so
so
NO
Kit
W)
I lours
24
24
24
24
24
24
24
24
24
24
12
12
12
12
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
21
24
24
24
24
24
24
12
12
24
24
24
21
21
24
21
21
Kate,
IJi./M.
.5
5
.5
1
1.5
1.5
3 75
2 5
3.75
2.5
1
,5
.5
.5
.5
1
1.5
1
1.5
1
1.5
1
1.5
!
3
1
3
1
3
1
3
1
3
1
3
1
3
1
3
1
3
1
1.5
1
3
1
3
3.75
2.5
1
3
1
3
3
1
3
lirom
Untreated
15, 16
10, 7
6,0
14, 13
7, 5
0,0
0,0
4,5
4,4
4,5
8,8
31, 32
14, 13
5,4
>
22, 27
5,4
1,2
1, 2
7,8
1,2
4, 4
4,5
3,3
4,2
6,7
36, 36
IK, 20
0,0
9, 10
10, 8
3 t, 37
-1, ft
ilk' liisidiifs, I'.I
Ire.itt-d (net)'
23, 2 1
32, 15
?S, 2"
32,41
21. 23
40, 51
28, 32
46, 44
16, 4 1
60, 52
79
V2
24
19
3, 1
7,7
3,3
6, 1
5, 7
10, 6
7,7
10, 10
0,0
0,0
6, 7
13, 36
4, 2
5, 8
10, 16
22, 2S
8, 16
31
7, 10
30, 32
8,9
37, 23
85,75
252, 190
24, 13
37, 65
11, 16
58, 30
31, 27
85, 73
6, 8
47,21
It, 22
50, 53
22, 29
»:,' 37
8!, 125
354, 338
11,13
60, 53
0
0
7, 3
0, 5
6, 7
\ 5
i, 1
i i, <•>
I/, ! 1
I s , c.f )
,M.
Sjr
^ 4
^
; ,
2J
2J
5'l
3i;
3l(
!
.«/
21
3f'
f\
I
.\
3
"
6
•»
0
f
6
0
J
1
!,":
10
,s.
1.'
«
10
bO
7 ,
i'.;
I/
14
I ^
2V
2 '
i
12
23
.'-.
.,.-;
II
i :
.
.;
i
\ ! •
; ;
3 i 'J
-------
TiiWc m.(Confimied)
Food
Veal loaf
liutkr
Olci >n"KtriiurHie
Shortening
Ik'fln, Spice's, UcvcMgos,, Mi>>c.
Cocoa
Colin1, ground
Brand A
Coliee, ground
Biaiul IS
CotFce beans
Rousted
Tea, green
T\n, orange pekoe
Allspice, ground
Cinnamon, ground
Ginger, ground
Nutmeg, ground
I'epper, red, ground
Yeast, dry
" Net = residue in tiealej — average
covered, J Extra fine gianulated. e Ble
package.
II.O, %
40
4.7
4.4
0.7
6.0
5 7
2,9
2.7
5,3
6.1
8.6
9.0
10.4
7.9
7.3
7.5
"f.
80
SO
SO
so
so
80
80
80
60
m
60
80
SO
80
80
80
80
80
80
80
80
80
80
SO
80
80
80
80
80
80
80
80
1 [ours
24
24
24
24
24
24
24
24
12
12
12
n
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
Rate,
I.U./M.
1
3
I
3
1
3
1
3
3,75
2 5
3,75
2 5
1.0
,j
.5
.5
.5
.5
1.5
1
1.5
1
1.5
1
1.5
1
1.5
Itrunmlc Kisicliits, I*. I*
I'nliciitrd
6,6
1, 1
3,3
1, 1
0,0
0, 0
1, 1
0,0
6, 6
7,8
4,4
4, 3
73,71
25,24
28, 28
1, 1
'1 rriilcd (ni'l)'
8, 8
27,25
6, 4
11. 1!
1.0
3, 7
I, I
2,8
47
33
25
29
11, 12
10, 10
4,4
5, 5
0,0
5,7
0,0
1,2
15,25
26,23
12,0
19, 16
8,8
6, 11
7,4
16, 11
9,9
6,5
0,0
0,0
.M.
Specifii
8
H 7
5
3 7
0.5
1,7
i
1.7
13
13
6.7
12
12
6.7
4
3.3
0
4
0
I
20
17
6
12
8
6
5.5
9 1
9
3 7
0
0
. i , , >> c- -c. i 3V« p p m. increase from fumigation - *•», ,
sidiK- in untreated sample. & Specihc residue ~ •• p j - , •/».*-• Cluxolutc
l,»t,*f"Uff'ni(T.i)fii-infi\l* *
ichcd. ' Bronuted. e Fancy patent. •* Enriched. ' Pastry flour.
, .. ,
tate ot UmuiMtion (11) M.)
m couiiucrctaf 6-02,
98 kg/ha. Crops of wheat grown on the soil the same year and two
succeeding years contained 0.42, 0.25, and 0.09% bromide in trie above
ground portions - first year plants which suffered scorching damage con-
tained as much as 0.61%. Otherwise similar soils containing 0.93% or
2.81% organic carbon retained 25 and 63 ppm bromide, respectively.
Dumas (1973) fumigated fresh fruits and walnuts with methyl bromide
and/or ethylene dibromide and determined the residues. Results are in
Tables 15-19. In general lower fumigation temperatures resulted in lower
bromide (Br ), but higher ethylene dibromide, residues.
-------
Reprinted with permission from j._ Agri.
Food Chem., 21:433-36 (1973). Copyright
by the American Chemical Society.
Table 15". Bromide Residues Found in Pulp and Skin ol Fruits Fumigated with Methyl Bromide at Various Temperatures, E»,
Normal Atmospheric Pressure (760 nun) lor 2 hr
Peach
Residues as bromide, ppm
Cherry
Temperature,
•c
25
25
25
25
21
21
21
15
15
15
10
10
10
4
4
4
Dosage motr-yl bromido,
mg/l
0 (control)
16
24
32
24
32
40
32
40
48
40
48
84
48
64
80
California
0.3
3.5
4.5
5.5
4.0
4.6
5.3
3.5
4.0
4.3
2,7
3.2
3.8
1.9
2.7
3.5
Ontario
0.2
4.9
4.2
3.2
2.3
3.9
2.4
4.7
California (Bing)
4.1
11,4
11.0
8.2
7.2
8.2
4.2
5.3
Caufornia.
(Schmidt)
30
6.7
6.1
5.9
4.6
3.5
4.7
Table It Distribution of Residue in Peaches and Plums (ppm)
Fumiyated with Methyl Gromide at Different Dosagus and
Temperatures lor 2 hr
Table <7- Residues of Ethyieno Dibrornirte and )»:>,-
in Apple;,'1 alter Fumigation with Ethytene Dibranndc
and 24 mg/l. ior 4 hr at 13°
Oosnge methyl bromide,
mg/l
0
16
Temperature,
Peaches
Peaches
Peaches
Peaches
Peaches
Plums
Plums
Plums
Fruit part
Skin
Pulp, outside half
Pulp, inside half
Pit v/all
Seed
Skin
Pulp
Pit
25
4,3
2,0
2.0
1.3
15.0
4.7
1.0
2.2
25
104
1.8
5.2
4.1
47.0
7,1
1 9
5.2
64
"C
4
3.4
1.9
2.3
1.1
15.7
8.6
1.8
6.2
Ethylene Usi.so-
miue residue.
ppm
36
14
4.5
1.2
75
40
13
1.6
0
" Apples (Delicious, variety) hrirt in cola Mor.uju
at 13" ,iti,.T tro.itrm nt " This includes sorno brotiiid
olhylono dibromide
Concentra-
tion, mg/l.
12
12
12
12
12
24
24
24
24
fumi^alior),
days
t
2
3
S
12
1
2
3
6
:, 4
Contiol nonfumigated apples
Table Ift, Residue of Ethylonc Qibromide and Inorganic Bromide
In Newly Harvested Macintosh Apples alter Fumigation with
Ethylene Dibromide 12 mg/l. for 4 hr at 13"
Concen-
tration,
mg/l,
12
12
12
12
12
12
12
12
12
Time after
fumiga-
tion, days
1
2
4
6
9
12
2
3
5
Storaqe
tempera-
ture. °C
13
13
13
13
13
13
25
25
25
Ethylene
dibromide,
ppm
23
3.6
1.2
0,17
0.14
0.23
0.2
0.15
0
Inorganic"
bromide
residue,
ppm
1.7
2.2
2.4
1.9
Control nonfumtrjatcKj
apples
0.8
• This inc!yj«» torno tiMimid'S rs'-yltmo from (ho eJhylonu dibromide
Table 14 Inorganic Bromide Residue In Shelled .ino "in-!
Walnuts nfter Exposure to Methyl Bromido ^t Various
Concentrations for 24 hr
Fumigation
tempera-
ture, °C
Without shell 25
25
21
15
10
5
2
~™ •*
™ A
In shall 25
21
15
to
5
CH.P'do- mi to
age, mn/l
0
16
24
32
40
58
64
64
64
tfi
?H
3?
4')
'.a
•m H10
Shell"
90
81
74
67
1 tO
12
14
17
•',21
-------
B. Environmental Transport
No specific information was found. When used in above ground appli-
cations, methyl bromide and ethylene dibromide evaporate into the yener-tl
atmosphere. When used in below ground applications, Tie thy 1 bromide has
to be "covered" to prevent rapid evaporation into the air, and ethylene
dibromide seems to stay very close to its point of insertion.
C. Bioaccumulation
ILartin et al (1956) grew vegetables and citrus seedlings in soil
treated with ethylene dibromide. In either a sandy loam or a silty clay
loam the citrus tops accumulated only about U.40% of Br over the range
1-12 ml ethylene dibromide per 3 gallons of soil; at the 0.5-0.8 ml/3
gal of soil level, accumulation was about 0.17%. Lima bean and carrot
tops accumulated more Br than the citrus seedlings, 1.35 and 0.60%,
respectively, at 8 ml/3 gal. Carrot tubers acquired only 0.10% at 8
ml/3 gal.
Munsey et al (1957, pp. 201-2) added 13 ppm of ethylene dibromide
to commercial bakers' flour and 20 ppm to rolled oats. Bread prepared
from the flour was free of unchanged fumigant. Boiling the oats in
water for one minute left 12 ppm of the fumiganl in the oats.
Young et al (1959) allowed cows to feed only on peanut vines grown
on ethylene dibromide treated soil. After 28 days of increasing Br con-
tent in the milk, these levels ranged 14-61 ppm from vines containing
48-314 ppm, respectively. The bromide levels were rising at a rapid rate
when the experiment was terminated.
Muns et al (1960) grew a variety of vegetables on earth treated with
ethylene dibromide at 4.67 g/m2. Lima bean straw contained L8, 76, and
J2?
-------
28 pptn Br when harvested 20, 19, and 16 weeks after soil treatment.
Corresponding sets of figures (ppm, weeks) for other crops were:
onion -9.1, 16
beet - 10.4, 10
turnip - 10.8, 8
spinach - 11.1, 8
lettuce - 14.9, 14
sugar beet tops - 18.3, 18
roots - 30.4, 16
shelled blackeye bean - — , 14 (none detected)
Lynn et al (1963) fed cows a ration consisting in part of methyl
bromide fumigated oats and corn. There was a direct correlation between
the Br content of blood and milk. Dietary levels of 10, 19, and 43 ppm
Br produced milk levels of 4-12, 7-12, and 10-20 ppm, respectively. The
Br levels plateaued at 4-5 weeks at the low and middle Br-in-feed levels,
and at 2-3 weeks at the high level. Milk contained a higher percentage
of Br taken in as a contaminant in the feed than as a NaBr dietary supple-
ment.
Getzendaner (1965) fed hens feed containing 50-410 ppm of Br from
methyl bromide fumigation. The following portions of the hens and eggs
were examined for Br content after 70 days (figures are average Br/Br in
feed, max. Br/Br in feed) :
light meat - 0.2, 0.23
dark meat - 0.3, 0.5
egg shells - 0.3, 0.5
skin - 0.4, 0.5
-------
liver - D.5, 0.7
feathers - 0.6, 1.5
kidney - 0.8, 0.9
egg whites - 0.8, 1.1
egg yolks - 1.2, 1.5
blood - 1.7, 2.1
Thus, except in the blood and egg yolks, the hen did not accumulate Br
over the amount it was taking in.
Wilson and Norris (1966) applied ethylene bromide to soil at 11 ml/m2
annually for nine years. Table 20 contains the Br content of the soil
and various crops grown on it in the last year. The figure for onions is
complicated by the poor growth of onions in this treated soil,, There is
no apparent correlation between the accumulation in the various root
crops.
Laue et al (1969) fed cows, calves, and piglets for 90 days on a
diet which had been methyl bromide-fumigated. A plateau was reached in
the blood and organs for a particular Br intake. The Br content of milk
and flesh was not hazardous for human consumption.
X. TOXICITY
A. Human-Occupational experience, Other
Prain and Smith (1952) discussed an occurrence in 1947 in which six
of eight boys died after exposure to methyl bromide from a fire extinguisher
in a confined area. Pre-death symptoms included convulsions, epileptiform
fits, and depressed reflexes. Massive pulmonary edema was evident, and
anuria became obvious within one day. The first urine passed by the two
survivors contained considerable amounts of albumin and many granular
32I4
-------
Reprinted with permission from Down to
Earth 22:15-18 (1966). Copyright by
Dow Chemical.
TABLE 20.
1til.il bfoniiiles m p p m. in various cropland Ihcinuck soil
in wliiili they were growing alter 9 years of treatment
with the same fuinig.ints. li>M data.
Sampling
Vegetable date EDB
Radlsh 7/20
Roots 378
Tops 665
Soil 16,5
Beet 8/2
Roots 2!6
Tops 469
Soil 16,5
lettuce 9/28
leaves 330
Soil 27
Carrot 9/29
Roots 45
Soil 27
Potato 9/29
Tubers 66
Soil 27
Onion 9/29
Bulbs 12
Soil 27
Celery 8/25
Stalks 402
Soil 18.5
Spinach 8/10
leav*» 102
Soil 18.5
casts; neither of the survivors showed pulmonary edema signs.
The authors conjectured that a toxic dose of methyl bromide caused
damage to the periphery of the respiratory system and to the renal tubu-
lar epithelium, and also caused cerebral upset.
Gallais et al (1952) discussed another case of methyl bromide leaking
from extinguishers in which one of three adults hospitalized later died.
All suffered from cerebral disturbances, dysarthria (speech difficulty)»
bilateral tnydriasis (dilated pupils), and swallowing problems. The
autopsy revealed extensive necrosis of the greater curvature of the stomarl
-------
hemorrhagic gastroduodenitis, brain congestion, and massive hepatic
fatty degeneration.
Kubota (1955) reported that human fatalities resulted from air con-
centrations of methyl bromide 1 600 ppm, but 100-150 ppra was harmless.
The blood of fatal cases had 211 mg/1 of Br, with survivors showing only
50 mg/1. Skin in contact with 8000 ppm developed pustules,
Winteringham and Barnes (1955) reviewed the symptomology of ire thy 1
bromide poisoning. There is a latent period following exposure even to
an eventually toxic dose. Headaches, dizziness, nausea, vomiting, weak-
ness, mental confusion, restlessness, mania, and finally tremors and
convulsions preceed death, usually from pulmonary edema.
Allen (1956) reported that there were no reported cases of fatalities
or even dermatitis from exposure to ethylene dibromide. Overexposure to
vapors produced irritation to the eyes, nose, and throat, headache,
giddiness, nausea; chronic overexposure damaged liver and kidneys. No
standard for maximum air concentration had been set, but the range 2-25
ppm was under consideration.
Turner (1958) reviewed the toxicology of di- and tetrabromomethanes,
recommending industrial exposures of < 25 ppm for CH2Br2 and f I ppm for
CBr^/8-hr.
Fiorentini and Mosinger (1958) described two fatalities from exposure
to 3 mg/1 of methyl bromide. Inflammation and degenerative changes in the
cerebellum, cortex, pallido-striatum, and thalamus accompanied cerebral
edema. Lower down, the heart muscle, kidneys, liver, and lungs exhibited
degenerative-inflammatory changes, hemorrhages, and stasis.
Franken (1959) discussed a fatality from chronic occupational ex-
posure to methyl bromide. Considerable damage nad occurred to the sensory
-------
and motor spinal roots and ganglia. Large lesions were present in the
cerebral and cerebellar cortex.
Kantarjian and Shaheen (1963) discussed eight non-fatal cases of
chronic occupational exposure to methyl bromide. They pointed out that
2-6 other workers similarly exposed were apparently unaffected. The
symptoms exhibited by the "eight" approximated the syndrome of
polyneuropathy - numbness and heaviness of the legs, all; unsteadiness
of gait, six; numbness of the hands, four; headache, coughing, anorexia,
aches, etc., two-three. Deep reflexes were absent or sluggish. There
were no severe systemic ill effects.
Drawneek et al (1964) commented that a serum level of 5 mg/100 ml
of Br in workers using methyl bromide occupationally could induce in them
a state of carelessness and euphoria not in keeping with the nature of
their work.
Collins (1965) discussed a new case of non-fatal occupational methyl
bromide poisoning and reviewed other cases, concentrating on the wide
variety of disorders resultant from damage to the central nervous system.
Mine (1969) discussed four fatal and six non-fatal occupational
methyl bromide poisonings occurring in California from 1957 to 1966.
Van Haaften (1969) reported the first known case of human poisoning
from acetylene tetrabromide, Br2CHCHBr2 . Hospitalization for severe
hepatic damage resulted from breathing vapors during one work day.
Apparently man reacts much more severely to this compound than do rats
(see the next section).
Araki (1971) reviewed 14 cases of methyl bromide poisoning in Japan
during 1964-1970. Tables 21 and 22 present the frequency of occurrence
of various signs and symptoms.
-------
Table 2L Symptoms of our fourteen cases
Table 22. Signs manifested in our fourteen cases
Symptom
'Gait disturbance
•Headache
'Numbness of the extremities
&)izzincss
Nausea, Vomiting
Speech disturbance
Blurred vision
Forgetfulncss
Irritability
Insomnia
Emaciation
Double vision
Chills, Shivering
Loss of libido
Depression
Anxiety
Asthenopia
Mumber
of cases
12
11
9
7
5
5
3
3
2
2
2
1
1
1
1
1
1
0S
/°
86
79
64
50
36
36
21
21
Sign
Ataxia of gait, Incoordination
Contracted visual field (n
Positive Romberg's sign
Exaggerated deep reflexes
Transient hypertension
Impaired superficial sensation
Hearing loss
Nystagmus
Muscular weakness
Hand tremor
Coma
Intention 'remor
Impaired deep sensation
Sluggish deep reflexes
Muscular atrophy
Inability to fix
Skin rash
Generalized convulsion
Pathological reflexes
lation
;d perimetry)
'S
ation
lumber
of cases
11
8
6
6
5
5
4
4
4
4
2
2
2
2
2
2
2
1
1
%
79
57
43
43
36
36
29
29
29
29
Sax (1968) gave TLV's and Toxic Hazard Ratings for a number of
bromohydrocarbons, as follows:
TLV (according to the ACGIH)
Methyl bromide 20 ppm (78 mg/m3)
Ethyl bromide 200 ppm (892 rag/m3)
Ethylene dibromide 25 ppm (190 mg/m3)
Toxic Hazard Rating
Methyl bromide Highest coxicity from acute local
ingestion, inhalation, and irritation,
also acute systemic inhalation and
ingestion.
Sub-fatal toxicity from chronic sys-
temic ingestion, inhalation, and skin
absorption.
-------
Ethyl bromide
Ethylene dibromide
Vinyl bromide
Highest toxicity from acute systemic
ingestion, inhalation, and skin absorption.
Sub-fatal toxicity from acute TocaJ
irritant, chronic systemic ingestion, in-
halation, and skin absorption.
Highest toxicity from acute loca L and
systemic ingestion, inhalation,, irritant,
and skin absorption,
Sub-fatal toxicity trom chronic local
irritant, chronic systemic ingestion, in-
halation, and skin absorption.
Sub-fatal toxicity from acute local
inhalation, acute systemic inhalation, and
chronic systemic inhalation.
Highest toxicity from acute local and
systemic ingestion, inhalation, and
irritation.
Sub-fatal toxicity from acute system!,
skin absorption.
Unknown toxicity from chronic local
or systemic contact.
From "Dangerous Properties of Industrial Materials" by N.I. Sax c 1975,
1968 by Litton Educational Publishing, Inc. Reprinted by permission of
Van Nostrand Reinhold Company.
B. Birds and Mammals
1. Acute, subacute
Porritt et al (1952) subjected meadow mice to atmospheres of 4 or 8
g/1 of methyl bromide; death occurred in three or two hours, respectively.
Rowe et al (1952) determined these LD-50's for ethylene dibromide
329
Propyl bromide
-------
vapor exposure (g/kg): 0.055 for female rabbits, 0.079 for chicks, 0.110
for guinea pigs, 0.117 for female rats, 0.146 for male rats, and 0.420
for female mice. The effects noted in rats included, most importantly,
lung irritation and hepatic injury; renal injury and central nervous sys-
tem depression were also present.
Smyth, Jr. et al (1954) reported an oral, range finding LD-50 of
75 mg/kg for 1,4-dibromo-2-butene in rats.
Valade studied inhalation toxicity of methyl and ethyl bromide on
dogs, guinea pigs, and rats. The LD-50's for 1/2-hour exposures were
(g/m3):10 for methyl, and > 100 for ethyl.
Davis and Hardcastle (1959) determined 24-hour median tolerance
limits of bluegill sunfish (Lepomis macrochirus) and largemouth bass
(Micropterus salmoides) for ethylene dibromide, ^5-18 and 25-50 ppm,
respectively (two sources of river water were used to hold the yearling
specimens used).
Balander and Polyak (1962) reported an LD-50 of 1.54 mg/1 for an
inhalation dose of methyl bromide in white mice.
Sokolova (1962) exposed rats to 1 g/m3 of methyl bromide. There
was a reduction in oxygen requirement from 3-4 m^/1 min. to 0.83-0.9
mg/1 min. Hemoglobin, erythrocyte and leukocyte counts lended to in-
crease, while serum catalase and cholinesterase tended to decrease.
Fuller and Morris (1962) introduced ethylene dibromide directly
into the crops of young pullets and old hens, with equivalent results.
Egg weight was reduced from a 1/2 mg/bird/day dose. Egg production
was reduced by an 8 (but not 4) mg/bird/day dose. Production ceased
from a 16 mg/bird/day dose. Recovery of production occurred 12 weeks
after cessation of dosage, but egg weight recovery required 6-10 months.
-------
Kutob and Plaa (1962) gave mice s.c. injections of di-, tri-, and
tetrabromomethane. No hepatic damage was seen from 29 mmole/kg of the
di-, 1.1 mmole/kg of the tri-, and only minimal from 0.05 mmole/kg of
the tetra-. Most suffered damage from 4.4 mnole/kg of the tri-, and
less than half suffered damage from 0.3 mmole/kg of the tetra-.
Kutob and Plaa (1962, pp. 354-61) reported these LD-50 values in
mice for a s.c. dose and a 10-day observation period (moiole/kg) :
dibromomethane - 21.5, tribromomethane - 7.2, and tetrabromomethane -0.9,
They also determined LD-100 for the tetrabromo-, 1.5 mmoles/kg (six of
seven mice died in 24 hours, the last in 48 hours). Based on the LD-50
values, the di- and tribromomethanes were classed as quick-acting, and
the tetrabromo- as delayed action. It may be seen from the preceeding
paragraph that a dose of the dibromo- greater than the LD-50 was still
not hepatotoxic in mice.
Hollingsworth et al (1963) reported LD-0 of 0.6 g/kg and LD-100 of
1.6 g/kg for oral doses of acetylene tetrabromide in rats.
Dykan (1964) reported that rabbits exhibited central nervous system
disorders from single exposures to 17-20 mg/1 of dibromomethane or to
11-13 mg/1 of tribromomethane.
Thompson (1966) reported a personal communication from H. A. U.
Monro of an incident in which two horses died after drinking water con-
taminated with 404 ppm Br from exposure to methyl broraide .
Institoris et al (1967) reported these i.p. LD-50's for male mice
(mg/kg): 1,4-dibromobutane, 300; 1,6-dibromohexane, 270.
Kakizaki (1967) reported a lethal dose for methyl bromide in rabbits
of 130 mg/kg s.c. Characteristics of poisoning were paralysis of the
hind limbs, cessation of drinking, and reduction of urine.
JJ1
-------
Leong and Torkelson (1970) reported an oral LD-50 of about 500 mg/kg
for vinyl bromide in male rats. Vapor toxicity studies showed 100%
mortality from 15 minutes exposure to 100,000 ppm (0.44 k^/m ;) and from
seven hours exposure to 50,000 ppm (no fatalities from 1 1/2 hours expo-
sure) . No fatalities resulted from seven hours exposure to 25',000 ppm.
2. Chronic
Rowe et al (1952) reported that guinea pigs, monkeys, rabbits, and
rats tolerated 25 ppm exposure to ethylene dibromide 7 h/d, 5 d/w, 24
weeks .
Rosenblum et al (1960) fed dogs for 6-8 weeks a diet which contained
35-150 mg/kg of Br from fumigation with methyl bromide. At the highest
level gross obesity and lethargy resulted. No interference with meth-
ionine metabolism or symptoms of Br intoxication were seen.
Balander and Polyak (1962) reported that exposure of mice; for two
hours a day for 30 days to 0.15 mg/1 of methyl bromide (l/10th of the
LD-50) had no cumulative effects. No effects at all resulted from 20
days exposure to < 0.01 mg/1. For rabbits this threshold concentration
was < 0.1 mg/1.
Dykan (1962) exposed rats for four hours a day for two months to
0.25 mg/1 of di- and tribromomethane. Both, especially the tribromo-,
caused disorders in the hepatic protein-prothrombin and glycogenesis
functions, and also in the renal filtration capacity. When injected in
100-200 mg/kg doses daily for 10 days, both compounds proved detrimental
to the liver and kidneys.
Kantarjian and Shaheen (1963) discussed a 1940 publication by D. D.
Irish et al. They exposed rats end guinea pigs to 0.25 mg/1 of methyl
bromide for 7 1/2-8 hours/d, 6 d/w, for six months without gross symptoms
332
-------
or histopathologic changes. Rabbits, however, showed paralysis of the
extremities after 14-16 days (non-permanent). Monkeys were unaffected
for at least five weeks, but some showed paralysis by three months (also
non-permanent). Apparently some of the rabbits and monkeys were not
affected at all during the course of treatment.
Thompson (1966) further elaborated on this publication by Irish.
All animals died from an exposure to 0.85 mg/1 lasting 12-24 hours.
Daily exposures to 0.42 mg/1 for eight hours was tolerated by rats for
one week-five month periods, but poor growth and intoxication resulted.
Guinea pigs were nearly unaffected after six months of the 0,42 mg/1
dosage. Rabbits showed severe nervous response after only a few days.
Even down to 0.13 mg/1 (but not 0.065 mg/1) the rabbits developed
paralysis.
Morris and Fuller (1963) demonstrated that ethylene dibromide had
a measurable growth depressant effect on chicks when given in their
diet at 1 40 ppm for one week.
Hollingsworth et al (1963) exposed a variety of animals to 14 ppm
of acetylene tetrabromide 7 h/d, 5 d/w, 14-15 weeks. There was no un-
usual mortality. Other series of exposures involved 4 ppm for 26 weeks
and 1.1 ppm for 28 weeks. The effects on growth, liver, and kidney
weights are given in Tables 23-26.
At the highest dose the lungs of all species except guinea pigs
showed signs of congestion, edema, and hemorrhage; rabbits and monkeys
were especially effected. At the middle dose female guinea pigs showed
all three effects, but only mice and male rats of the other animals
showed any lung troubles. The low dose was uneventful medically.
33:
-------
TAHI F. 23.
Final Average Body, Li\cr and Kidney Weights From Rats that Received Repeated Seven-hour
Exposures to Acetylene Tetrabromide Vapor
Cone.,
Controls
14
Controls
14
Controls*
4
Controls*
4
Controls
1 I
Controls
1 1
No. of
10
10
8
10
1R
15
18
17
17
16
19
20
Sex
M
M
F
F
M
M
F
F
M
M
F
F
No. of
0
70
0
70
1-7
127
128
128
181
131
132
132
on
100
100
100
100
180
180
181
181
190
190
191
191
Av«.
WU, g
322
2H7d
195
1R4<>
34H
3H9
209
191
38fi
3118
234
237
Organ Weight*
Liver
g
7 50
8 35-1
4 89
5 77"
7 4S
8 25J
5 25
5 78'
8 52
8 60
5 64
5 90
g/lOOg
2 33
2 93"
2 50
3 14-
2 15
2 4,1-
2 51
2 93-
2 21
2 35
2 48
2 42
Kidney
t
2,. 12
2:. 07
1.53
] 515
2 .19
•2 35
1 .48
1 54
2 24
2 14
1 .52
1 .52
K/lOOg
0 66
0 73
0 78
0.85
0 63
0 70
0 71
0 7S
0 58
0 58
0 68
0 69
*Air-) P = <0.001 lh) P=0.01 (•) P-0.05
(*) P->0.06
TABLE ZM.
Final Average Body, Liver and Kidney Weights from Guinea Pigs that Received Repeated Seven-hour
Exposures to Acetylene Tetrabromide Vapor
Vapor
Cone.,
ppm
Controls
14
Controls
14
Controls*
4
Controls*
4
Controls*
1.1
ControU*
1.1
No. of
Rats
8
S
8
8
8
g
8
Sex
M
M
F
F
M
\1
F
8 . F
8
8
7
6
M
M
F
F
X
[
*
t
1
*
1
I
•-
1
No. oC
Exp.
0
73
0
73
129
129
130
130
133
133
134
134
Days
on
Expt.
101
101
101
101
182
182
183
183
192
192
195
195
Final
AVK.
Body
Wt.. g
861
72O
811
673'
926
744-
862
7K5
852
807
856
815
Organ Weights
Liver
E
25 16
26 61
25 94
24 53
29 55
24 02
28 31
25 62
26 5
26 48
29 66
28 75
g/lOOg
2 92
3 70-
3 20
3 64"
3 20
3 24
3 29
3 27
3 14
3 28
3 47
3 53
Kidney
E
1. 94
-1 86
i> 74
4 36
!> 92
!> 41
5 45
4 96
'> 84
5 14
a 79
1 74
g/lOOg
0 69
0.68
0.71
0.65
0 64
0 73*
0.63
0 63
0 69
0.64
0 68
0 58
*Air-exi>os«'d controls
**This \st-itfht is an uvcraRp value for u group of two i
(•) H-.<0 1)01 t.1') l'-0 004-0 006
mimala
(') P-0 03
* TABI.B ZS.
Kinal Average Body, Liver and Kidney Weiffhts from Female Mice that Received Repeated Seven-hour
Exposures to Acetylene Tetrabromide Vapor
Cone.
Control
14
Control*
4
Control*
1 1
No of
10
9
9
7
9
«
No. of
Exp
0
73
127
127
130
130
on
105
105
180
180
189
189
Final
Avg.
Body
Wt.,g
32
31
35
31-
27
27
Organ Weights
Liver
K
1 92
2 lot
1 92
1 73
1 51
1 44
g/100E
6 0
6 9'
5 5
5 6
5 6
5 4
Kidney
t
0 42
0 41!
0 5i!
0.4!)
0 34
o 3;:
K/lOOs
1 3
1 3
1 5
1 5
1 3
1 2
'Air-exposed controls
(') !'->0.1 {>•) P-0 048
(•) P=<0 001
-------
TABLE 26.
Final Average Body and Organ Weights from Rabbits and Monkeys that Received Repeated Seven-hour
Exposures to Acetylene Tetrabromide Vapors
Vupor
Cone.
ppm
Control
U
Control'
4
Control1
1.1
Control
U
Control
1.1
No. of
Anirn
g/lOOg g
1 86
2 90
2.36
2 95
2.68
2 42
2 14
2 53
2 32
2.52
12 8
14 3
15 6
15 8
14 2
17 6
19 6
21 6
16 3
19 8
g/lOOg
0 41
0.46
0 40
0 42
0.42
0 39
0 42
0 40
0 34
0 43
Testes
K
g/100B
5 97 j 0 22
4 09
5 98
5 12
4 79
5 51
8 55
4 22
—
0 14
0 16
0 16
0 13
0 13
0.18
0 08
—
-~
•R-Rabbits
•*M = Monl
-------
No statistically significant (at p 1 0.05) external or internal changes
resulted which were also dose-related.
3. Sensitization
4. Teratogenicity
5. Carcinogenicity
6. Mutagenicity
7. Behavioral effects
No mention was found in the literature of findings bearing on
3, 4, 5, or 6. Many of the bromohydrocarbons seem to have a lethargy-
inducing property in man and animals. Most of the incidents of serious
human exposure to methyl bromide mentioned alterations in behavior from
brain damage.
C. Lower animals
In Table 27 is a compilation of scientific names in alphabetic order
of insects and worms for whom there was found in the literature some in-
dication of toxicity from bromohydrocarbons. Where the scientific name
was accompanied by a common name, the latter was included in the table.
Also in the table is the specific bromohydrocarbon, and the growth stage(s)
of the insect if known. Because in most instances the toxic effect: was
an intentional one, and in some reports it was not clear what, was meant
by "toxic effect", and considering the general difficulty of determining
percentage kills in large soil or stored products samples, it; was decided
not to further elaborate on most of the reports from which the table was
compiled. The first reference to a particular insect - bromohydrocarbon
combination is identified in the table, along with year of publication,
by the "control number" assigned to the reference. Some of these refer-
ences were secondary, the actual work having been reported prior to 1952.
-------
Table 27. Insects Known to Be Susceptible to
Bromohydrocarbons
Acanthoscelides obtectus (bean weevil); MeBr, EtBr2; 10757 (1954)
Acarus siro (cheese, wheat mite); MeBr; 10793 (1966)
Achroia grisella (lesser wax moth); EtBr2, 12155 (1958); EtBr, 14709 (1965)
Aedes aegypti (yellow-fever mosquito, eggs); EtBr2; 12589 (1962)
Agriotes (wireworms); EtBr2; 10717 (1956)
Amphimallon majalis (European chafer, all stages); EtBr2; 11331 (1962)
Ancylostoma caninum (canine hookworm, larvae); EtBr2; 12653 (1954)
Antagenus piceus (black carpet beetle); MeBr; 11318 (1962)
Anthrenus flavipes (carpet, furniture beetle, all stages); MeBr; 11318 (1962)
Anthrenus verbasci (varied carpet beetle); MeBr; 11318 (1962)
Aphelenchoides ritzema-bosi; 1,4-dibromopropyne; 11402 (1958)
Aphelenchus avenae (nematode); EtBr2; 11199 (1971)
Argas persicus (tick); MeBr (EtBr not effective); 15335 (1955)
Ascaridia galli (chicken worm); 1,1-dibromoethane; 12355 (1952)
Ascaridia lineata (chicken worm, eggs); MeBr; 10734 (1955) i
Atta cephalotes (ant); MeBr, EtBr2; 10739 (1955)
Balaninus elephas (larvae); MeBr; 11047 (1954)
Baris lepidii; MeBr; 14944 (1965)
Belonolaimus (sting nematode); EtBr2; 10734 (1955)
Belonolaimus longicaudatus Rau (sting nematode); MeBr; 11333 (1962)
Brachycerus (weevil); MeBr; 12491 (1963)
Brachytrupes membranaceus (DRU)(cricket); EtBr2; 10747 (1954)
Calandra granaria (grain weevil); EtBr, PrBr, BuBr, PeBr, HexBr; 12936 (1953)
Caloglyphus krameri (mite); MeBr, EtBr2; 14883 (1970)
'337
-------
Garpomyia vesuviana (Costa) (fruit fly, eggs,, larvae); EtBr2; 10718 (1955)
Geratitis capitata (Wied.) (Medit. fruit fly, larvae); EtBr2; 10719 (1956)
Chilo agamemnon (corn borer, larvae); MeBr; 11172(1970)
Jochlicella barbara (snail); MeBr; 14237 (1965)
^onoderus amplicollis (Gyll.) (Gulf wireworm); EtBr2; 10722 (1953)
Conoderus falli Lane (southern potato wireworm); EtBr2; 11259 (1966)
:Jonoderus vespertinus F. (tobacco wireworm) ; EtBr25 11259 (1966)
Oonotrachelus nenuphar Herbst (plum curculio, larvae) ; MeBr, EtElr25 11257
(1966)
Cryptolestes ferrugineus Stephens (rusty grain beetle); MeBr; 17849 (1967)
Cryptolestes turcicus Grouvelle; MeBr; 17849 (1967^
Dacus cucurbitae (Coq) (fruit fly, eggs, larvae); EtBr2; 10718 (1955)
i»
Dacus dorsalis - see write up following table (1954)
uacus ferrugineus (Fab.) (fruit fly, eggs, larvae); EtBr2; 10718 (1955)
Ijacus zonatus (Saund) (fruit fly, eggs larvae); EtBr2; 10718 (1955)
Dendroctonus engelmanni Hopkins (engelmann spruce beetle) ; EtBr;? ; 10723
(1953)
Dendroctonus monticolae Hopkins (mountain pine beetle); EtBr2; 10575 (1955)
Dendroctonus pseudotsugae Hopkins (Douglas fir beetle); EtBr2; 10575 (1955)
Ditylenchus destructor (potato rot nematode); EtBr2; 10734 (1955)
Ditylenchus dipsaci (eelworm); EtBr2; 12706 (1958); MeBr, larvae, 11409
(1959)
Dorylaimus (nematode); MeBr; 11128 (1972)
v
Dyspessa ulula (Borkhausen) (carpenterworm moth); MeBr; 12491 (1963)
Ephestia elutella (tobacco moth, all stages); MeBr: 13415 (1959)
-------
Ephestia klihniella (Medit. flour moth, larvae); MeBr; 12343 (1952)
Galleria mellonella (greater wax moth); EtBr2; 12155 (1958); EtBr,
14709 (1965)
Glyptotermes dilatatus (live-wood termite); EtBr2; 14812 (1970)
Gnorimoschema operculella (potato tuber moth); MeBr; 13728 (1958)
Helieotylenchus; EtBr2; 10698 (1956)
Hemicycliophora parvana Tarjan (sheath nematode); MeBr; 11333 (1962)
Heterakis gallinae (from poultry, eggs); MeBr; 10734 (1955)
Heterodera avenae (cereal eyst-nematode); MeBr; 15766 (1970)
Heterodera glycines (soybean eyst-nematode); MeBr; 12928 (1958)
Heterodera marioni (rootknot eelworm); MeBr, EtBr2| —
Heterodera rostochiensis Wollenweber (potato root eelworm, golden nematode
of potatoes); MeBr; 12278 (1952); EtBr2; 11054 (1953)
Hoplolaimus tylenchiformis (Daday) Andrassy (lance nematode); MeBr;
11333 (1962)
Lampetia equestris (narcissus bulb fly); MeBr; 13486 (1952)
Lasioderma serricorne (cigaret beetle, adults, larvae, eggs); EtBr2;
12916 (1958)
Laspeyresia splendana (larvae); MeBr; 11047 (1954)
Leptinotarsa decemlineata Say (Colorado beetle); EtBr2; 11241 (1971)
Limonius agonus (eastern field wireworm); EtBr2; 11053 (1954)
Matsucoccus resinosae (scale of red pine); EtBr2; 13425 (1959)
Meloidogyne hapla Chitwood (root-knot nematode); EtBr2; 10698 (1956)
Meloidogyne incognita var. acrita (root-knot nematode); EtBr2; 10756 (1954)
Meloidogyne javanlca (root-knot nematode); MeBr; 13399 (1959)
•j-ic,
39
-------
Musca domestica L. (house fly, larvae); EtBr2; 11418 (1962)
Nippostrongylus muris (larvae); BuBr; 10585 (1955)
Ophiobolus graninis (take-all); MeBr; 15766 (1970)
Oryzaephilus surinamensis (sawtoothed grain beetle); MeBr, EtBr?; L0757
(1954)
Ostrinia nubilalis (corn borer, larvae); MeBr; 11172 (1970)
Oulema melanopa (cereal leaf beetle); MeBr; 14887 (1970)
Panagrellus redivivus (nematode, pre-adult); 1,4-dibromopropyne; 11402
(1958)
Paratylenchus (pin nematode); EtBr2; 10559 (1961)
Periplaneta americana (American cockroach); EtBr2; 14204 (1964)
Phylloxera vitifoliae - see write up following table (1962)
Pleocoma (fruit root grub); EtBr2; 17851 (1970)
Popillia japonica (Japanese beetle, grubs); EtBr2; 14553 (1958)
Pratylenchus penetrans (root-lesion nematode); MeBr, EtBr2; 14569 (1961)
Pratylenchus pratensis; MeBr; 12935 (1953)
Pratylenchus vulnus (root-lesion nematode); EtBr2; 10756 (1954)
Quadraspidiotus perniciosus Comst. (San Jose scale); MeBr; 17843 (1967)
Radopholus similis (burrowing nematode); EtBr2; 13931 (1961)
Rhagoletis mendax (blueberry maggot); MeBr, EtBr2; 14349 (1970)
Rhagoletis pomonella (apple maggot); MeBr, EtBr2; 12619 (1962)
Rhyzopertha dominica (lesser grain borer); MeBr; EtBr2; 10757 (1954)
Rotylenchulus reniformis (nematode); MeBr; 15188 (1960)
Sitophilus granarius (granary weevil) ; MeBr; 13476 (1952); Et.Br2; 10757 (1954)
-------
Sitophilus oryzae (rice weevil); MeBr, EtBr2; 10757 (1954)
Stegobium paniceum (drugstore beetle); MeBr, EtBr2; 10757 (1954)
Syngamus trachea (from poultry, eggs) ; MeBr; 10734 (1955)
Tarsonemus myceliophagus (mite); MeBr; 17838 (1966)
Tenebrio molitor (larvae, adults); MeBr; 13759 (1959)
Tenebroides mauritanicus (cadelle, black grain gnawer); MeBr; 13476 (1952);
EtBr2; 14197 (1961)
Theba pisana (Muller) (white garden snail); MeBr; 14237 (1965)
Tribolium castaneum (red flour beetle); MeBr; 14243 (1965)
Tribolium confusum (confused flour beetle); MeBr, EtBr2; 10757 (1954)
Trichodorus christiei Allen (stubby-root nematode); MeBr; 11333 (1962)
Trichostrongylus axei (nematode); MeBr; 15811 (1965)
Trichostrongylus colubriformis (nematode); MeBr; 15811 (1965)
Trogoderma granarium (khapra beetle); MeBr; 15091 (1952)
Tylenchorhynchus martini Fielding (stylet nematode); MeBr; EtBr2; 10699 (1956)
Tylenchulus semipenetrans Cobb (citrus nematode); MeBr, propargyl bromide;
10772 (1966)
Tyrophagus (lintneri) (mite); MeBr; 14488 (1962)
Tyrophagus putrescentiae (mite); MeBr; 12310 (1966)
Xiphinema index (dagger nematode); MeBr; 15506 (1971)
Zabrotes pectoralis (Mexican bean weevil); MeBr, EtBr2; 10757 (1954)
Cockroach; MeBr; 15130 (1961)
Fly; MeBr; 15130 (1961)
Pink bollworm (cotton); MeBr; 12776 (1952)
Termite; MeBr; 15130 (1961)
-------
MeBr - methyl bromide, EtBr - ethyl bromide, EtBr2 - 1,2-ethylene dibromide,
PrBr - n-propyl bromide, BuBr - n-butyl bromide, PeBr - n-pentyl bromide,
HexBr - n-hexyl bromide
Hinman (1954) tested the effectiveness of a variety of compounds
against day-old eggs, and third-instar larvae of the oriental fruit fly
Dacus dorsalis. Exposure time to the vapors was two hours at 24°C.
Results are in Table 28.
Reprinted with permission from J. Econ.
Entomol. 47:549-56 (1954). Copyright by
the Entomological Society of America
Table 23. Effectiveness of various compounds usud as fnmtgants against naked CRRS nnd tliirfl-
instar larvae of the oriental ftutt lly. Milligrams per liter giving 50 and 95 percent mortality 48 hours
after eiposure.
COUPODNU
//ntt>gt ittihtl .Wtiihnfjf! j
Bromides
Methane, dibrotuo- (methylene dibromijK/l bromide)
Hiitauc, 2-bromo-J-inctlijl- (l-amyl liroiuidc)
Butane, l,?-*!ilir£*mo-
Uutane, l,3-rniuu-
l^iitanc, 2,l-dibi»niO"
Hexane, l-liroiuo- (n-hexyl brnmidf)
HeiiUK-, l-bromu-*i-t;tli> 1-
Hrxanr, l-l>r«iiio-3,.'>,5-triinelhyI-
llvxane, 2,5-iHbroiiio-
Heptuiif, 1-biuuio- (n-lieptyl bromide)
Heptane, ° broino- (Vr-liepHl broinMe)
Octane, 1-bronio- (ii-.wtjl bromide)
Octane, J-liriiiiin- (*rr-tn tji broiniite)
Nonnne, 1-bruiiio- (ii-ininjl bromide)
Detanc, l-broaio- ('i-decyl brojnide)
Dmlernne, 1-liroino- (H-dodecyl briiuikk')
Tetrudecaiie, broino-
Ilcxadecane, broino-
LD-so
'lyilmearha
IB
43
>2.1U
HI
>iW
> 151
Si
>1W5
>1SH
0.1
17!>
>1KS
>IR7
>l";i
>1!H
>IK;l
i.i
>1HI
411
>170
S8
> IK'S
>I7U
>Ua «
>15«
>llfl
>1U7
>105
^ l.ill
Hi
>IW
u
>111
> 110
>10U
>1IHI
>07
>««
LD-iw
w, Xatunital
38
1'JII
>2.M
HU
>ir.i
> 151
yj
>l!)j
>1SS
18
5.5
>17i>
>1KH
>1S7
> I7:i
>!9t
>tna
8.7
>INI
>IM
>170
81
> IKi
>170
?!-;
>ISfi
>ll!l
>1H7
5- HW
> IM
>I1«
>ito
il
>111
>ll(l
>UH1
>1(M»
>I>7
>!»li
f.v
LI Mil
132
>S05
75
2U1
>?I3
>151
HS
>ins
Kit
0.3
VA
-J
31'
*«>
48
>10I
>1KI
4,1
14 5
21
3. a
44
88
O.C
> 17 1
>15«
Mia
>!<("'
> IIU
> l^U
40
>H3
>11S
>H1
>lin
>109
>ll«
>U7
>90
IIVM.
1.1JIW
fiH
>ao$
>8,»0
>.1(N1
>i!3
> 151
im
>11>J
>1H8
1.8
in
130
<15'
... 131
>I1S
>10l
>183
7.8
SI
4S
>no
94
>l(i2
l.B
>l?t
>15ti
>llll
>1U7
>10a
> ISO
>H8
>143
>11S
>in
>110
>109
>100
>87
>M
' Mortality \W >14«
I'M >1!U
rnali tl Cyt t,ij,,ir<,/,ni
-
a
:• I'M
III
>I4fl >l*6
ifl
7.3
>':!«
> m
tl
-------
For comparison the author used these data from Balock and Lindgren
(1951): methyl bromide had LD-95 for eggs at 25, and larvae at 19 mg/1;
ethylene dibromide had LD-95 for eggs at 0.8, and larvae at 0.6 mg/1.
Monro et al (1961) in 1953 began a study on two wild and one labora-
tory strain of the granary beetle Sitophilus granarius which involved treat-
ing them with methyl bromide vapor and breeding new generations from sur-
vivors of > LD-50 (or higher) doses. Exposure was standardized at five
hours, 25°C, and 70% R.H.; only adults were used. The results are in
Figures 6 and 7. The "A" selected strains were begun after 1956 from
survivors of > LD-50 doses, whereas the selected non-A strains were sur-
vivors of > LD-75 doses; apparently the wider gene pool available to the
"A'"s increased resistance faster. The non-selected groups were the con-
trols for the experiment, and showed no inherent ability to increase re-
sistance. Discontinuance of selection did not cause reversion to the
1953 resistance level. At the time of writing, the LW strain had shown
a 24% and the MW strain a 41% increase in body weight. Simultaneous ex-
periments with Tribolium confusum and Tenebroides mauritanicus did not
generate much increase in resistance.
An A strain which was 5.5 times more resistant to an LD-50 dose of
methyl bromide as the normal was also shown to be 3 times more resistant
to an LD-50 dose of ethylene dibromide.
Rammer and Stafford (1962) exposed first-instar female nymphs of
Phylloxera vitifoliae (Fitch) to a variety of brominated propanes for
four hours at 21°C, or for eight hours at 21°C in the presence of soil.
Some studies were also done at 13 and 30°C. The results are in Tables 29-
33 and Figure 9.
-------
Selection suspended
Non-selected (normal strain)
15 20 25
Successive generations
FIHI>I.(\K Mo /Lb
l.S-nilinmici- 0 tl a
l.i,S-'l'rilui,nifi- 1 ti li
1,9-1)
!,-M>
1-llro
•'-I
() ^
In, .11.1, :i ililoro- 1
III OHIO- H
!!., It
" 1'. V iMiiirs' exposi'i t'
^n'T. .rtit tinTcrences In
!).r) <•
1 .1
5 o
no soil
t \\ccn i
romi'im! \.iLjrs Ji.ive no ]i'McM in
0 It I.MUI
- - \ VI .11
Micro- S,
niolcs/i.. A
2 IB
/i.oi;
H ?,"i
.','t *t
r . ^ i [rl> 1C Deo
1'' > l M. I. Sl,Ol'F
01!' 0 ."'S 12 (i
.V, 1 il-i '1 (I
1" ¥ dS !> '!
1 L< 'i ] xi ,j 't
117 !) 1 (ll'l 1') 7 10 II
le.ms nt tlic 1* o 'c.cl nre ni(!,,-iile*l \vlien
,,,nii)ion. DuiK.ma .Mn!|];.lo lijinjre Test
Figures 6 & 7 reprinte.
with permission from
Ana. Appl. Biol. 49:37
377 (1961). Copyright
the Association for
Applied Biologists.
Lindgren and Vincent (1962) studied the effect of moir.ture content of
various commodities on mortality of Tribolium confusum and Sitophilus
oryzae from application of a fixed dose of metliy] bromide. '['he results
-------
Table 30.-Effect of temperature on the toxicity of 1,3-
dibromopropane to grape phylloxera nymphs. (4 hours' ex-
posure without soil.)
TK.MTMi-
M-rnr
r !•'•)
").)
70
8.5
KDsn
Mo./i..'
0 . .")0
O.H
(i :!8
Ri i, \riVK
YAPOH
S VTl U VI K»M
AT Kl),n
o . o;i(i
0.01!)
0.010
F,nw
Me /i,.
O.fifl
0..5.5
— tj
SLOPE
10 7
12.6
'2.0
a Vi Mpnifif .ml r'rflcrrnrc U'lncrn rnc.in offer I:no rlo^cs nt t.ie 5% Jc^rl.
DiHH.in's Mulliitlc ll.uifc l<",t (l'IV>).
'' l)l> " is m.i !cp"i!<>il o\\mtf to tlif 'Inrrj.'(_-nce of U'i Inchest romentMtion
fruni tin- Kcm-r»l IVTIK! •-*-* U\ ttie oilier llirec conrctitriidon-*. The KD-.o ;md
hlnpr .TTC ha'-oil on tlic llnre !o\\c*-t conr rntr.tfions.
Table 31. Toxicity of various bromopropanes to grape
phylloxera nymphs.'
K.|),nvmHT G l'.l>.»
1)111 son, is Mr; /H7
G DHV
PitopA\Kh M(,.c Mirromoles SOIL SLOPE
l.-l-Dilirorno S (ij a '28 0 B.OO 17 «
1-llromn- '20 1 I) IS') 4 50 * S.*
1.2-Dilironui- 'ifi 1 e 1CT.3 .11..'. l^.J
],^-l)ihrt>mo-:l cliloro- ?OHI.O '1 Hl)'10,0 :12.>0.0 6.4
•l 70° F , H hours' exposure. 100 grains soil of 15°^ moisture.
'' Tlie i;i)-,ii lor l.^,'i-tnl>rnni».pron;ine was ffreittcr than 15.H8 mg.
c hu:nifu-rtnt (I'lTerrnces between menps sit tlie 1% level are indicate.'! when
compnreil \aines lia\e no letlors in common. Punrnn's Multiple Rnnge Test
(III.;.-.).
Table 33.. -Effect of soil temperature on the toxicity of 1,3-
dibromopropane to grape phylloxera nymphs."
TKMITIM-
T( UK
<° !•'.)
.5.5
7(1
8,5
KI),n IN"
Mc./S7(;.
Ditv .Son,1'
1 1 .50 a
a (i.5 1)
1.2-2 r
KD™ IN
Mo./87 ("..
I)u\ Son,
16 20
0.66
1.8-2
SlOPF,
26.7
17.6
7.7
1 8 hour-.' exposure, 100 pr.uns vil of I.'icp moisture.
Slpniflf .int (IitTpiemrs t ctuccn menus ;it tl'2 1 ^t levi'l nrp indicate'! whcn
cnnipareil \alurs lm\c no Ictlcri in ciniimon, J)iincanS Multiple H:inge Test
(lil.05).
Table 33.-Effect of soil moisture on the toxicity of 1,3-
dibroniopropane to grape phylloxera nymphs. (8 hours' ex-
posure, 7(T F.)
Moiiiiiu: KDon iv KUW iv
COMIM" Mo./ST ci. Mo./87 G.
(%) DHY Sou.-1 DRV Son,
.5 (ir. (i.Gti 17.(i
2.58 3.13 15.5
H KIHout ;>'/<, moisture i^iitcnL ('tl.O grama) was greater than 5'JtG mg.
b KIU g. .soil
c KID -i g. soil.
•/ Duiuan's Multiple Rnngc '1'est (IDj.i) imiicnlej a algniticant ditfereni-e be-
Uct-ii iiK,ini at tlie 1% Ie"rl
Tables 29-33 reprinted with permission from J. Econ. Entomol. 55:203-
11 (1962). Copyright by the Entomological Society of America.
-------
are in Table 34. Regardless of commodity or moisture content, 100% kills
of T. confusurn were obtained if the concentration (actual) x time value
(CT) was greater than 75 or if at least 50% of the applied dose (CL-50)
remained unabsorbed by the commodity for four hours; corresponding values
for S. oryzae were: CT over 31, and CL-50 over 2.5 hours.
Moje (1963) tested CH3(CH2)nCH2Br, n = 2-9, against citrus nematode
larvae. He found that toxicity increased by a factor of 2.45 for each
additional CH2 group. Cyclohexyl and cyclopentyl bromides were less toxic
than the n = 2 compound.
Harein and Soles (1964) tested crotyl bromide (86% l-bromo-2-butene,
14% 3-bromo-l-butene) and 1,2,3-tribromopropene against the adults of
Tribolium confusum, Oryzaephilus surinamensis, Lasioderma serricorne, and
the larvae of Attagenus piceus. The results are given in Table 35.
Reprinted with permission from J. Econ.
Entomol. 55:203-11 (1962). Copyright by
the Entomological Society of America.
CONCENTRATION IN MG 1_. (LOG SCALE!
Fid. 1. Klfect of temperature on (lie mortality of grnpe pliyl-
i
-------
Reprinted with permission from J. Econ.
Fu
Entomol. 55:674-78 (1962). Copyright by
the Entomological Society of America,
Table 3U.
— Concentrations found ;iiid mortalities of adults of Tnbolium confusum and Silopliilus ortjzae obtained v h<"\
•ni^ltmj vimous tommodiues >tt different moisture contents with methyl bromide in 10-liter recircuialion tluni'it rs
i»f« • 24 hows at 70° F. Load: 75%.
~ - - —
.MulNl'I !!!•:
CON MM1
COMMODITY tVt'l
B4*l*}' " " °
12.0
15. «
SKta i.!r.il „„, «!„...•
t, X' '.t 1 I',.
Ml VN
A\Miuji ''
("ONI i NIK \-
•i ION >'M<; /I )
7.1
5. i
5 0
6. -2
5.7
-JMJ
-5 8
4 1
-2 !)
O v>
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-------
Reprinted with permission from J. Econ.
Entomol. 57:369-70 (1964). Copyright by
the Entomological Society of America.
Table *£ -Toxicity of 2 chemicals to 4 species of stored-product insects fumigated 24 hours at 80° ±4° F. and at a
relative humidity of 78 + 18S, in 19.5-liter bottles.
Do-agc (nig/l)
Ll)aii fiducial limit* LDa fiducial limit-.
low High LDso LJHV Uijj
CimlWii fl.mrbi-rllo •> (l.Mt O.H'Ji 0
Satt-loiillut) gi.iin Inn-tie «) 17 -<:i I.Sj1'
Ci^uvt«. lu-i-llt- .:!.» ,;H .47 51
liliick <-nriH-l i.i'i-tk' l.IS 1 01 1.35 •> UU
Cr,ilull>ri 1 i" 1 3N 1 ill
1 7«l i D7
] (H» 1 OH ! 'Kl l.H 1 59
Black carpet l«vlk- S !W 3 5.» + a3 «15 S.ll 7tt
D. Plants
Table 36 is similar to Table 27 dealing with toxicity of bromohydro-
carbons to plants and fungi rather than insects. Many of the entries re-
sulted as incidental findings from studies of insect toxicology. Refer-
ences to delayed germination of seeds, and fruit damage were omitted,
Table 36. Plants and Fungi Known to be Susceptible to
Bromohydrocarbons
Alternaria solani; MeBr; 17879 (1959)
Armillaria mellea (citrus root rot); MeBr; 15637 (1969)
Aspergillus parasiticus; MeBr; 14731 (1970)
Botrytis cinerea (soil fungus); MeBr; 12252 (1953)
Ceratostomella fimbriata (sweet potato black rot); EtBr2; 10734 (1955)
Chenopodium album (fat hen); MeBr; 10782 (1954)
Colletotrichum atramentarium (soil fungus); EtBr2; 13492 (1952)
Corticium solani (soil fungus); EtBr2; 13492 (1952)
Cyllndrocladium scoparium (pine root-rot); MeBr; 11093 (1971)
Cyperus compressus (annual sedge); MeBr; 11420 (1962)
-------
Cyperus esculentus (yellow nut sedge); MeBr; 11420 (1962)
Cyperus rotundus (nut grass, Topalak weed); MeBr; 15657 (1969)
Digitaria sanguinalis (crab grass); MeBr; 11420 (1962)
Fusarium bulbigenum lycopersici (tomato wilt); MeBr; 14334 (1968)
Fusarium lini (soil fungus); EtBr2; 13492 (1952)
Fusarium oxysporum var. auriantiacum (soil fungus); EtBr2; 13492 (1952)
Fusarium oxysporum f. niveum (soil fungus); MeBr; 17785 (1954)
Fusarium vasinfectum (cotton wilt); EtBr2; 10734 (1955)
Gallium aparine (goose grass); MeBr; 11420 (1962)
Gallium asprellum (rough bed straw); MeBr; 11420 (1962)
Hemileia vastatrix; MeBr; 11094 (1971)
Lepidium sativum; MeBr; 14885 (1969)
Lepidium virginicum (Virginia peppercress) ; MeBr; 10782 (1954)
Linaria canadensis (blue toad flax); MeBr; 11420 (1962)
Mycelia sterilia; MeBr; 14885 (1969)
Orobanche ludoviciana var. cooper! (broomrape) ; MeBr; 13775 (1959)
Orobanche ramosa (broomrape); MeBr; 13732 (1958)
Oxalis latifolia; MeBr; 14722 (1964)
Panicum repens (torpedograss); MeBr; 12562 (1963)
Penicillium rubrum; MeBr; 14731 (1970)
Phytophthora cactorum (soil fungus); EtBr2; 13492 (1952)
Phytophthora cinnamomi (soil fungus); MeBr; 12252 (1953)
Jh?
-------
Phytophthora citrophthora (soil fungus); MeBr; 12252 (1953^)
Phytophthora cryptogea (soil fungus); EtBr2; 13492 (1952)
Phytophthora fragariae (strawberry red stele disease)- MeBr; 13017 (1957)
Phytophthora parasitica var. nicotianae (tobacco black shank); MeBr;
12879 (1956)
Plasmodiophora brassicae (cabbage clubroot); MeBr; 13457 (1960)
Polygonum aviculare (wireweed); MeBr; 10782 (1954)
Poria hypolaleritia (tea root-rot); EtBr2; 11092 (1969)
Pythium ultlmum (soil fungus); EtBr2; 13492 (1952)
Rhizoctonia solani (soil fungus); MeBr; 17784 (1953)
Saccharum spontaneum (grass); MeBr; 10748 (1956)
Sclerotinia homeocarpa (soil fungus); MeBr; 12252 (1953)
Sclerotinia minor; MeBr; 14885 (1969)
Sclerotinia sclerotiorum (soil fungus); EtBr2; 13492 (1952)
Sclerotium bataticola; MeBr; 13399 (1959)
Sclerotium delphinii (soil fungus); MeBr; 17784 (1953)
Sclerotium rolfsii; MeBr; 10738 (1955)
Solanum opacum (black nightshade); MeBr; 10782 (1954)
Spergula arvensis (spurge); MeBr; 11420 (1962)
Synchytrium endobioticum; MeBr; 14145 (1970)
Thielaviopsis basicola (black root-rot); MeBr; 13459 (1959)
Tilletia foetida (wheat bunt); MeBr; 14335 (1968)
Trifolium glomeratum (cluster clover); MeBr; 10782 (1954)
Urocystis tritici (wheat flag smut); MeBr; 14336 (1968)
-------
Verticillium albo-atrum (soil fungus); EtBr? ; 13492 (1952)
Verticlllium dahlias (soil fungus); EtBr2; 13492 (1952)
Waitea circinata (pine root-rot); EtBr2; 17850 (1971)
Beans (plants); l,4~dibromo-2-butene, l,4-dibromo-2-butynej 11426 (1957)
Beets (seeds); EtBr2; 10749 (1955)
Broccoli (seeds); EtBr2; 10749 (1955)
Carnation; MeBr, EtBr2; 12931 (1953)
Carrots (seeds); EtBr2; 10749 (1955)
Celery (seeds); EtBr2; 10749 (1955)
Clover (seeds); EtBr2; 10749 (1955)
Corn (seeds); EtBr2; 10749 (1955)
Cucumbers (plants); l,4-dibromo-2-butene, l,4-dibromo-2-butyne; 11426 (1957)
Eggplant (seeds); EtBr2; 10749 (1955)
Gladiolus; MeBr; 10738 (1955)
Groundnut; MeBr; 10701 (1955)
Lettuce (seeds); EtBr2; 10749 (1955)
Lime; MeBr; 10762 (1954)
Maize (plants); l,4-dibromo-2-butene, l,4-dibromo-2-butyne; 11426 (1957)
Morning glory (plants); l,4~dibromo-2-butene, l,4-dibromo~2-butyne; 11426 (1957)
Mushroom; MeBr; 17838 (1966)
Mustard (seeds); EtBr2; 10749 (1955)
Narcissus; MeBr; 14236 (1965)
Nutgrass; MeBr; 10727 (1955)
Oats (seeds); EtBr2; 10749 (1955)
Oats and wild oats (plants; l,4-dibroiao-2~butene, l,4-dibronio-2-butyne;
11426 (1957
-------
Onion (seeds); EtBr2; 10749 (1955)
Orange; MeBr; 10762 (1954)
Pea (plants); l,4-dibromo-2-butene, l,4-dibromo-2-butyne; 11426 (1957)
Potato; MeBr; 12273 (1952)
Radish (plants); l,4-dibromo-2-butene, l,4-dibromo-2-butyne; 11426 (1957)
Rutabaga (seeds); EtBr2; 10749 (1955)
Rye (seeds); EtBr2; 10749 (1955)
Rye (plants); l,4-dibromo-2-butene, l,4-dibromo-2~butyne; 11426 (1957)
Spinach (seeds); EtBr2; 10749 (1955)
Tobacco (seeds); MeBr; 16482 (1957)
Tomato (seeds); EtBr2; 10749 (1955)
Turnip (seeds); EtBr2; 10749 (1955)
Cobb (1956) reported that susceptibility of seeds to methyl bromide
generally increased with moisture content; temperature ?nd exposure time
/ere also factors. Even though some seeds survive and germinate, the
resultant sprouts may be weak and die soon or produce stunted plants.
Martin et al (1956) found that orange seedlings absorbed Jir from
soil treated with ethylene dibromide. Concentrations of Br in the leaves
of 0.17, 0.33, 0.40, 1.3, and 1.8% produced growth reductions of 12, 22,
31, 57, and 90%, respectively. Leaf Br concentrations of 2.5 or 1.5% in
carrots or lima beans, respectively, were not deleterious to growth.
Whitney et al (1958) studied the toxic effect of fumigation with
methyl bromide on barley, corn, grain sorghum, oats, and wneat seeds.
They found that little or no injury resulted when all of the following
conditions existed: seed moisture < 12%, dosage < 32 kg/m3, exposure <
24 hours, and temperature = 27°C . Relative tolerances of the seeds
examined were: oats > barley > grain sorghum - corn -• \heat.
t',?
-------
Viel and Giban (1958) found that an application of 200 g/m2 of ethylene
bromide, the usual for nematode fumigation, was harmless to the growth of
tomatoes. Retardation resulted from a dose 10 times that amount.
Blackith and Lubatti (1960) reported that moisture content of seeds
was a greater factor in damage from methyl bromide fumigation the greater
the oil content of the seeds. The oil also increased germination delays
by storing the fumigant in solution,
Blackith and Lubatti (1965) reported the results of a six year study
on germination ability of seeds containing an 8-18% water content after
fumigation with 0-1200 mg/1 for one hour (or equivalent) of methyl bromide.
Their results are in Table 37,
Table 37.
FtKenlage geimituiliou cafmcity (means 0/400 seeds levied on each occasion) of fumigated, stored cereals
Dosage of methyl bromide in mg.h./l.
Cereals
(IVko)
"Wheat (Allc)
Oils {Star}
Oats (Blenda)
Biriey (Procter)
Bittey (Herta)
Rye (Winter)
MUM (Wz68)
Moisture
content.
%
8
II
M
18
8
ii
li
IS
8
ii
M
18
8
II
14
IB
8
ii
»4
18
8
II
»4
18
8
ii
M
18
&
ii
— 14
18
Contru
Is
(ui)fuinigatcd seed)
Storage
3 years 0
93-o
89-5
88-5
31-8
95'°
91 -8
92-8
87-0
36-5
84-5
0-5
98-0
99"3
96*0
48-8
96-5
96-5
95-0
o
97'5
99'3
95-5
4'3
9J-8
99-o
69-5
a3'3
—
—
—
—
for
years
91-3
87-0
90-5
«4'5
93'°
85-0
88-3
o
85-0
82-0
65-0
o
96-8
97-8
96-0
7-8
95-3
94-0
93-8
o
93'5
96-5
84-7
o
28-0
2:7.5
4'3
0
94-8
94-0
73-0
o
(ConcGiitiation
600
Storage
for
3 years 6 years
92-0
94'5
24-3
6-8
93-0
92-0
40-5
0-3
85-8
82-5
60-3
7'3
98-5
94 '5
89-0
3-0
97-3
95-8
81-5
5a-8
99 '»
99'3
75'5
o
98.3
93 >o
39-o
23-8
—
—
—
—
93-3
93-0
25-0
o
9*-"5
88-5
35-8
o
80-0
77-0
46-8
o
96-5
94-5
86-0
o
97'S
94 '»
8 '"3
17-8
93-8
92-0
67-8
o
37-8
13-5
0-8
o
96-5
65-5
3* '3
o
x time pioduct)
1200
Storage
for
3 years 6 years
93 •<>
,85-0
25-5
I'O
96-0
63-5
41-8
3-5
82-0
69-3
36-5
i-3
96-3
97'5
75'5
1-8
97'3
96'3
81-8
0
98-3
81-3
75'3
o
97 '3
7«'3
44 -8
—
—
—
—
88-8
90-0
20-8
o
92-0
58-0
30-5
o
76-0
69-0
23-3
0
97-0
93-°
55-3
o
95-3
92-0
85-0
o
94 '5
83-8
7°"3
o
35'7
9-3
o
o
96-8
48-0
a -7
o
353
-------
Wilson and Norris (1966) applied ethylene dibromide to a soil each
year for nine years at 11 ml/m2. Average crop yields for the last three
years as a percentage of the yields from an untreated soil were: onions -
64, potatoes - 44, carrots - 102, celery - 106, beets - 83, lettuce - 79,
radish - 113, and spinach - 92. The reduced yields of onions and potatoes
resulted from poorly growing plants, not smaller sized "fruit".
E. Microorganisms
Table 38 is a listing of microorganisms reported in the literature to
have shown some susceptibility to bromohydrocarbons.
Table 38. Microorganisms Known to Be Susceptible
to Bromohydrocarbons
Agrobacterium tumefaciens; MeBr; 11344 (1962)
Bacillus anthracis; MeBr; 13475 (1952)
Bacillus subtilis; MeBr; 13118 (1966)
Coccidia; allyl bromide, 1,3-dibromopropene, l,4-dibromo-2-butene;
12951 (1952)
Escherichia coli; MeBr; 13118 (1966)
Fanleaf-yellow Mosaic Virus; MeBr; 15506 (1971)
Pseudomonas angulata (Angular spot); EtBr2; 10747 (1954)
Pseudomonas tabaci (Wildfire); EtBr2; 10747 (1954)
Pseudomonas tomato; MeBr; 17785 (1954)
Rhizobium trifolii; MeBr; 17785 (1954)
Salmonella paratyphosus A, B; MeBr; 12939 (1952)
-------
Salmonella typhosus; MeBr; 12939 (1952)
Shigella dysenteriae; MeBr; 12939 (1952)
Staphylococcus aureus; MeBr; 13118 (1966)
Tobacco Mosaic Virus; MeBr; 11299 (1962)
Vibrio cholerae; MeBr; 12939 (1952)
Xanthomonas veslcatoria; MeBr; 17785 (1954)
XI. CURRENT REGULATIONS
The following collection of foods and bromide residues permitted in
them was obtained from the Federal Register through 1967; there had been
only one change in the decade preceeding that year. No explanation for
the different bromide ion tolerances from methyl bromide and ethylene
dibromide treatment of the same food was found. When a food has been
treated with both, the higher tolerance is used.
Table 39. Allowed Bromide Residues in Foods
Treated with Bromohydrocarbons
Food Methyl Bromide Ethylene Dibromide Oth_er£
ToleranceS Tolerance8
Alfalfa hay
Apples
Apricots
Asparagus
Avocados
Barley
Beans
Beans, green
50
5
20
100
75
50
50
50
10
50, a
-------
Beans, lima
Beans, snap
Beets
Broccoli
Cabbage
Cantaloupe
Carrots
Cauliflower
Cereal grain,
milled fractions
Cheese,
parmesan
roquefort
Cherries
Cipollini bulbs
Citrus citron
Cocoa beans
Copra
Corn
Corn, forage
Cottonseed
Cucumbers
Dog food
Dried apples
apricots
50 5
50
30
75
50
20
30 75
10
125 125
325 325
325 325
20 25, b
50
30
50
100
50 50, a
50
200 25
30 30
400
30
30
25
25
356
-------
Dried dates 100
eggs C C
figs 150
peaches 30
pears 30
Eggplant 20 50 60
Garlic 50
Grain sorghum
(milo) 50 50, a
Grapefruit 30
Grapes 20
Hay, timothy 50
Horseradish 30
Jerusalem
artichokes 30
Kumquats 30
Lemons 30 30
Lettuce
Lines 30
Litchi fruit 10
Mangoes 20
Melons 75
Melons, honeydew 20
musk 20 40
water 20
357 .
-------
Nectarines 20
Oat flour
Oats
Okra
Onions
Oranges
Papayas
Parsnips
Peaches
Peanuts
Pears
Peas,
b lackeyed
with pods
Peppers
Pimentos
Pineapple
Plums
Popcorn
Potatoes,
sweet
Processed foods
not already
covered as of
6-15-66
d
50
30
20
30
20
30
20
5
50
50
30
30
20
20
240
75
75
125
d
50, a
50
75
25
30 25
40 25
25, b
50
50
125
35B
-------
Processed grains
for fermented
malt beverages
Processed herbs
and spices
Prunes
Pumpkins
Quince
Radishes
Raisins
Rice
Rutabagas
Rye
Salsify roots
Soybeans
Squash,
summer
winter
zucchini
Strawberries
Sugar-beets
Tangelos
Tangerines
Tons toes
Turnips
20
20
5
30
50
50
30
50
30
200
30
20
20
30
30
30
30
20
30
50, a
50, a
50
25
50
40
-------
Wheat 50 50, a
Yams 30
a - no limit on organic bromide
b - total of organic and inorganic bromides
c - 400 from a mixture or from nethyl bromide alone
d - 200 from a mixture
e - 125 from a mixture
f - a mixture of methyl bromide and propargyl bromide, in ppm of inorganic
Br
g - in ppm of inorganic Br unless otherwise indicated
XII. STANDARDS
No information was found.
360
-------
LITERATURE CITED
Alon, A., D. Ader, B. Bernas, and N. Levite (1962). The determination of
tetrabromoethane (TBE). Bull. Res. Council Israel, Sect. C. 11:225-34.
14672
Alumot, E. (1972.). The mechanism of ethylene dihroraide action on laying
hens. Residue Rev. 41:1-11. 16546
Alumot, E., and Z. Harduf (1971). Impaired uptake of labeled proteins
by the ovarian follicles of hens treated with ethylene dibromide.
Comp. Biochem. Physiol. 39(lB):61-68. 16226
Alumot, E., arid E. Mandel (1969). Gonadotropic hormones in hens treated
with ethylene dibromide. Poultry Sci. 48:957-60. 16225
Amir, D., and R. Volcani (1965). Effect of dietary ethylene dibromide on
bull semen. Nature. 206:99-100. 14131
Araki, S., et al (1971). Methyl bromide poisoning: a report based on
fourteen cases. Japanese J. Ind. Health. 13:507-13. 16086
Balander, P. A., and M. G. Polyak (1962). Toxicological characterization
of methyl bromide. Gigiena i Toksicol. Novykh Pestitsidov i Klinika
Otravlenii, Dokl. 2-oi (Vtoroi) Vses. Konf. 1962, 412-19. 12232
Barduhn, A. J., H. E. Towlson, and Y-C. Hu (1960). The properties of gas
hydrates and their use in demineralizing sea water. U.S. Dept. Comm.,
Office Tech. Ser., P B Rept. 171,031, 69 pp. 11889
Barnsley, E. A. (1964). The formation of N-acetyl-S-(2-hydroxypropyl)-
L—cysteine from 1—bromopropane. Biochem J. 93:15p. 16864
Barnsley, E. A., T. H. Granby, and L. Young (1966). Biochemical studies of
toxic agents. The metabolism of 1- and 2-bromopropane in rats.
Biochem J. 100:282-88. 11244
Berck, B. (1965). Determination of fumigant gaues by gas chromatography.
J. Agr. Food Chem. 13:373-77. 14656
Bieloral, R., et al. (1965). Determination of ethylene dibromide in
fumigated feeds and foods by gas liquid chromatography. J. Sci. Food
Agric. 16:594-96. 16583
Blackith, R. E., and 0. F. Lubatti (1960). Influence of oil content on the
susceptibility of seeds to fumigation with methyl bromide. J. Sci. Food
Agric. 11:253-58. 15142
Blackith, R. E., and 0. F. Lubatti (1965). Fumigation of agricultural
products. XX. Prolonged storage of cereals fumigated with methyl
bromide. J. Sci. Food Agric. 16:455-57. 14233
361
-------
Bray, H. G., and S. P. James (1958). formation, of mercapturie acids from
aliphatic compounds in. vivo. Bioehem J, 69:24p. 12743
Bray, H. G., and S, P. James (1960). Metabolism of 1-bromobutane.
Mochem J. 74: 6p. 10537
Bray, H. G., J. C. Caygill, S. P. James, and P. B. Wood (1964). Formation
of mercapturic acids. V. Metabolism of some halogenoparaffins and
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16610
-------
Dumas, T. (1962). Determination of 1,2-dibromoethane in air and as residue
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363
-------
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3614
-------
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-------
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16125
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Munsey, V. E. (1957). Residues in foods and feeds resulting from fumigation
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369
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Federal Register Regulations
No specific reference was made to these, but data were taken from all
of them for the completion of the bromide residue tolerances given in
Section XI.
Tolerances for residues of inorganic bromides (on agricultural commodities)
from soil treatment with ethylene bromide. Fed. Regist. 21:768(Feb. 3, 1956)
10576
Tolerances for residues of inorganic bromides from fumigation with methyl
bromides. Fed. Regist. 20:9822(Dec. 21, 1955) 10578
Exemption from requirement of tolerances for residues of carbon disulfide,
carbon tetrachloride, ethylene dichloride, and organic bromide residues from
ethylene dibromide; tolerances for inorganic bromide residues from ethylene
dibromide. Fed. Regist. 21:5620(July 26, 1956) 10687
Food additives. Inorganic bromides. Fed. Regist. 31:8369-70(June 15, 1966)
10780
Food additives. Inorganic bromides. Fed. Regist. 31:12841(Oct. 1, 1966)
11256
Inorganic bromides: tolerance for residues. Fed. Regist. 27:8070-74
(Aug. 14, 1962) 11324
Inorganic bromides. Tolerances resulting from fumigation with methyl
bromide. Fed. Regist. 27:4623(May 16, 1962) 11345
Inorganic bromides; tolerance for residues. Fed. Regist. 26:12249(Dec. 22,
1961) 11401
Tolerances for residues of Inorganic bromides resulting from fumigation with
methyl bromide. Fed. Regist. 23:1365, 5465-66. (1958) 12156
Tolerances for residues of inorganic bromides from soil treatment with
ethylene dibromide. Fed. Regist. 23:4002(June 7, 1958) 12157
Tolerances for residues of inorganic bromides in or on litchi fruit after
fumigation with ethylene dibromide. Fed. Regist. 23:2966(May 2, 1958)
12393
Food additives. Fumigants for grain mill machinery. Fed. Regist. 28:6916
(July 6, 1963) 12606
Food additives. Fumigants for processed grains used in production of
fermented malt beverages. Fed. Regist. 32:7911-12(June 1, 1967) 13068
Inorganic bromides resulting from fumigation with methyl bromide. Fed.
Regist. 32:7173(May 12, 1967)
Tolerances for residues of inorganic bromides resulting from soil treatment
with ethylene dibromide. Fed. Regist. 22:4384-85 (June 18, 19')7) 13375
-------
Tolerances for residues of total combined bromine in or on cherries and
plums after fumigation with ethylene dibromide. Fed. Regist. 23:6553-54
(Aug. 23, 1958); 23:6665(Aug. 28, 1958) 13724
Inorganic bromides resulting from fumigation with methyl bromide; tolerances
for residues. Fed. Regist. 30:2104(Feb. 16, 1965) 14184
Food additives. Inorganic bromide. Fed. Regist. 29:3394(Mar. 14, 1964)
14208
Inorganic bromides resulting from soil treatment with combinations of
chloropicrin, methyl bromide, and propargyl bromide; tolerances for
residues. Fed. Regist. 30:7385-86(June 4, 1965) 14724
Inorganic bromide resulting from soil treatment with ethylene dibromide;
tolerance for residues. Fed. Regist. 30:14101(Nov. 9, 1965) 14954
Inorganic bromide; permitted residues from fumigation with methyl bromide.
Fed. Regist. 25:8368-69(Sept. 1, 1960) 15192
Tolerance for residues of inorganic bromide. Fed. Regist. 25:8948-49
(Sept. 17, 1960) 15194
Food additives. Futnigants for grain-mill machinery. Fed. Regist. 29:4672
(Apr. 1, 1964) 14209
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EDTA
SUMMARY AND CONCLUSION AS TO DEGREE OF HAZARD
EDTA and its various Na, Ca, and other metal salts is being
produced in fairly large quantities. The major uses dc not degrade
the organic portion of the compound and would appear to be ultimately
resultant in its release to the environment. Ordinary sewage treatment
does not degrade EDTA. Soil does not appear to retain EDTA, but can be
"weathered" by it and also be subjected to minerals exchange.
There appears to be little danger to humans and domestic animals
from oral ingestion because of the very low absorption from the
gastrointestinal tract; only chickens show ability to absorb and
metabolize EDTA. Plants readily abosrb EDTA into their roots, differ
widely in ability to transport it, and show ability to metabolize it.
Plants also vary widely in tolerance to the EDTA which they have absorbed.
Insufficient information is available on toxicity to aquatic (including
marine) life to arrive at any conclusion about the hazard of EDTA to
these segments of the biosphere.
-------
EDTA
I. PHYSICAL PROPERTIES
Wendlandt (1960) ran differential thermal and thermogravimetric
analyses on EDTA and two of its common salts, Na2EDTA and Na2CaEDTA, as
obtained from various American suppliers. The results of his (values
in this type of analysis are somewhat dependent upon the instrument and
operational technique employed) DTA studies are given in Table 1. EDTA
is seen to decompose at 250 or 265°, Na2EDTA at 230-294°, and Na?CaEDTA
at 337-403°. In Table II are his TGA results, which indicate the manu-
facturer may over or underdry the hydrated salts. Wendlandt thought he
had demonstrated the possibility that the calcium salt was a mixture of
1- and 3-hydrates, rather than the 2-3'H20 previously suspected.
Bhat and Iyer (1967) ran TGA's on the following EDTA's:
BaH2'4H20, BiH'H20, CaH2-2H20, CoH2'3H20, CuH2-H20, DyH-2H20, NiH2'H20, and
SbH. All of these decomposed before melting except Bi (292°), Cu (238°),
and Sb (290°) , the latter three decomposing just over their mp's. The
Bi water of hydration was not liberated until after decomposition had
set in, indicative of its being bound to the metal atom. The order of
thermal stability found was Dy > Sb > Bi > Ni > Cu > Co > Ca, Ba; this
order did not correlate with the stability constants or heats of forma-
tion in solution.
Koechel and Frank (1966) reported that: EDTA was soluble in
alkalis, ethanol, ethyl ether, and chloroform, slightly soluble in water,
and very slightly soluble in most other organics; Na2EDTA was soluble in
water to about one part in eleven (and gave a pH of 4.5 as a 0.1 M
aqueous solution), but just barely soluble in organics; Na2CaEDTA was
37')
-------
soluble in water on a one to one basis (and gave a pll of 7,5 as a
0.1 M aqueous solution). The sodium salts of EDTA exhibit the order
of solubility in water: tetra > tri > di > mono. The dissociation
constants of the four protons are: 1.0 X 10~2, 0.2 X 10~2, 6.3 X 10~7,
5.0 X 10~n, indicating that EDTA is a stronger acid than acetic.
Nomenclature of the EDTA's is complex, the term EDTA itself being
indiscriminately applied to the tetraC-COaH) and di(-C02H)-di(--C02Na)
forms. Presented below is a collection of names compiled by the
Chemical Abstracts Service of the American Chemical Society and pub-
lished in Desktop Analysis Tool for the Common Data Base (1968).
Table I. Results of Thermal Analysis of EDTA and its Derivatives
(Minimum thcrmogrovimctrie decomposition tcmpi-rntureB)
Temp.,
Compound Transilion ° C,
EDTA (./. 1'. Jiaker) liDTA -* doooinjro-ition 2.00
t!)T \ i Si IIIH -I rear AA) KDTA — * nij)uMti:i>TA 21I.O — Xiv-EDTA 11-1
Nu.EDTA — Nu.Cf), 23('i
X«. KDTA. 211-0 (J. T. Baker) Na.KI)TA.2H.() -. Xa.UDTA 1 Id
Kti-EDTA -» N:i.(.'Oj 2.V.
Na-EDTA 2H 0 (J. T. Baker) (3° Na-EDTA 2HJ)-> X.iJ-'DTA IOC,
C. per mm.) Na.KDTA — Xii/'Ch 230
Xa.Cui:i)TA 2-;m;U (Sequestrcne Xu-CaliDTA sU o —• 37
Xa''Ca) Na.CaHl>TA.:iH O
Na.CaKIJTA.Hll.0-* 85
CaCO,
Xa,CaEDTA.2-3H,0 (Scqwstrone Na-CaEIJTA jrlLO -*• -18
\V2Ca) (3° C. por min.) Xa.CaKDTA .HILO
Na-Ci.ni/r A :?u o-» r,3
Xa.CaKin'A HI 0
Na.CaliDTA lll.O -* 123
_ XasCalCDTA
Endotherm I'cak Maxima
EDTA (J. T. Baker) KDTA — iJ<-cr>ni|i i.-itiun
KDTA (Seqiiesirvnu AA) KDTA -*• ucoom|«;i.-!tion
NaIEDTA.2H1O(J. T. Baker) Na-lCDTA -'lf,.O -» Xa,.iJlJTA
X:t»Kl)TA — * tl«'t( m|)0?Hion
Xa2EDTA.21I50 (Eastman) Xa>:i)T\.2lI.O -» X:i.,r.l>TA
Xa-KI/1'A -* iVroinjvii-itwu
X«iEDTA.21LO (5'equest/ene Xa2) X-t.J-JDTA 211 ;U -* \j,LI)'J'A
Xu.KDTA -*
Xa.CnEDTA :iH.O -* K1S
Xa-CaKDTA UI.O
Nn.CaK1)TA 111 O — 19D
_Xa:CaKnTA
NnCaKl)'J'A — » tit r'imjwsiUon H1H
-------
Table II. Weigh* Loss Data for EDTA
and Ifs Derivatives
Water, %
Theo-
Corn)K)\i]«l Experimental rctirnl
Nn.EDTA-2U,0 10 5 9 08
(J. T. Baker) 10 2
9.7
10 1
K;I;EUTA-211*0 10 ! 9 68
(E:i.-tnmn) 10 0
N;i..]C]>TA-L'l!.,O !> H 9,(18
1 7^ (residual
\vutt-r)
l <>:\
I fii
11 ;' (3-hy- 13.1,2
11 8 '
12 fi
Table Ila. Synonyms for IOTA and its Salts
Nz')0rlu'i,,.','Y.,
fu-i-tic ,iold, ( i-t hyl .-ncdtn Itr Uo) tt'tra-, tctrasodlum salt
ft<\u,imal Un Kt RC K
Coi^ot fTRrr.
Ci'lon i: IfCHVJ
Colon H Ittt'.TN
c.-ton n; irmr*)
ftn-.^lox SU'
Ccnirjon BC ITC1TN
Df'itol 8 MtHCK
Rialol
Edaihanll tetra*>odtum ftBt
EDTA, sodium salt CAHF
EDTrt tctrasodiuu salt KKRCK, IECMTM
Endrate t«:tran"dium MERCK
Ethylcncb$s>l iwJno Ji ac«t Ic ncid] t ct rasodlum salt HEOCK
N,N'-Ettiy lent di.if i n«*diacctic aeid tctraaodlum salt IRCMTH
Et hi/l«snrdi«niriot« Iraace t Ic acid, te t ra sod i uin salt ADI , MEKCK, CFR
( lltliylKfiedl I. i tri lo )tet raacet ic acid tetraaodlum salt MERCK
Irgalon HLRCK
Ka'lo* IECMTfJ,MfhCK
Komplexon M'.RCK
Hetaquest C 1ECMTH
K'ervanald B NLHCK
II u 1 lapon HPHCK
Nultajion BF-12 ITCMTN
Mul Upon Ur-7« 1F.CHTM
Mullapon ETC Cone 1KCMTN
Hullapon BI'C Cfj I U in *• t h1./ I r n* (' i ;ni IIP If t r.1 n <' *> ( I r <"*r i d
f.odlnm -loM of r t >. / I • ni-tl t *mi nr t >• \r «<>< •- 1 I 0 arid Jf.CMVN
!.v "(.••• I.'.,
Tables I and II reprinted with permission
from AMUJ±!)e»k> 32:848-50 (I960).
Copyright by American Chemical Society.
-------
Tot racci-i I n Mf.RC K
Tc t rnaodi um EDTA II'CMTN
Trt rasoili um <• t h v 1 <: nod i a m i nr; 11: i r a ace t a t e 1 ECMTN ,CFR , US AH
Te t Til ')od i um c t h ;/ 1 onrct i a n 1 no t r t r ace t a t o
Tc traaodium ( <-•1 !iy 1 en <'d i n i t r i 1 o ) t e t r a ac e t n t c: USAN
Tetrasodium 33 \ t KU1A IIX Ml N
Tc tr.isodl uin salt of EDTA ICCHfM
Tetra.iodlum 9 ,11 t of <• t hy 1i: m-d i dm i nc t c t r ace t 1 c acid IECMTN,VBB
Tetrinc MFHCK,1ECMTN
Trllon 13 MERCK, I ECMTN
TST
Tycl arojol MEfifK
Verscno 67 IECMTN
Versene 100 IFCMTN
Veracne lit PCK
Verseini Dedds IECMTN
Versene FT :i ADT
Verseno Powder IECMTN
Warkt. clatr H-'t^ lECT'.rN
Wnrke-n>-.1 i n i tri lo)tctraacet Ic acid
Mptaqucst A IECMTN
Nervanaid b acid IECMTH
Nullapon IS acid IECHTM
Nullapon tIF acid IEC11M
Pcrina Klucr f.O acid IECMTN
Sequestrcne AA IECI1TN
Scqucstrlc acid IECMTN
Sequestrol IECMTN
Tet r1nc Acid
Trllon B, Trllon BW
Tri Ion BW
Veraene
Verscne acid IECMTN
Warkeelatc Acid IECMTN
Acetic acid, (cthyIcned1n11rilo )tetra-, trlsodlum salt
EDTA trlsodium salt IECMTN
Ethy lened i ami neacet Ic acid trisodlun salt MERCK
Perm/i Mcer 50, trisodtum salt IECMTN
Sequeatrene trlaodlum
Sequestrene trlaodium salt MERCK
Tri Von AQ IECHTN
Trl3odium cdetnte USAN
TrUodlum EDTA IECMTN
Trlsodlum hydrogen c i !
-------
10,j.a
Acetic acid, ( ft h yl e n ed i n I t r i '. o )tc t ra- , disodlum salt
Complexon III IECMTN
Dtsodium due id ctlvylom-dianlnetetra acetate
Disodiuri dihydrogcn c t hy I c-nod i *m ( nc t c t r aaect at e FCC
Di sodium e dot ate UGA ,M, USP, USP- A
Ditodium E!>TA I CCMTM ,FOC ,CFR ,MDE, ADI
1)1 sodium e t hy 1 nu-di in i ni t et r jacc t at e f CC , USP , VDB ,CF R
U I sodium c t li" li'iii'd (ami IV to t tvace t i c acid MDE
Uisodium ( ft tiy li nodi n i t r i lo )t ol r aacctat «• USP
Di sod in in ( e t hv icn.-d i n i I n 1 o) IP tr iiare t I c acid
Dliodium s.itt of I.Ill A IECHTN
Oisodtiidt soq in- n t r me C/XHt"
F> i 'tod i u n v»* r y r nc
Fil.ith.imll rii-ioJiun ADI.TARK
Ldut.ite Dlnodluu API
i'DTA
E.nrA, di-jo.Mnm ;i(i 1 t
TDTA di vodi nni CAKK
rndrnlr il i -.od i u-n H'M! ,ADI ,CART
I lhylon«-dj i• t r n.icot at r, disodium salt CAHP
!' t !•,'.; 11 iu-d i unl n, U' t r,«rtcc t ic ar. id, dliodium 3nlt AI>I
H.- t.i-i n. 11 il II I'M N
Pern, a M i i- r MJ c r w -i I li l,vdrogon v. t hy t <-nedt am luet c t raacet a te ditiydrote FCC
Uisodium IDTA dthydrate ICCMTN
Dliodlura c t!", lni.-1i.imf i\.tc traacctatc dihydrate FCC.IF.CMTN
KDTA di^o.Jin,' d. iiydra t •• CAPF
licqiipstreni U\2 It'CMTN
At'itic jcid, ( ctNyli n:- 1 c i u 11 ^ .- r y v^iio I c CAHK
EDTft Ci.lciiin salt CMU'
C, 0Mlft..'( a
Acetic <-cid, ( cttiyl enrUIn i tr I lo )t«t ra-i dicalclum salt
Ca-HDTA
Calc iur, rt>TA
Calcium cthy1rncdiamine tetraacctate CAHF
Calcium t^trt1.ccmin
Dt C«l c tmn FDT A
F.DTA, c.ilcium silt CARF
C«C, 0H, , ,?,Na
Acetic acid, ( ct hy I encd i n i t r I lo )t et ra-, calcium disodium solt
Calci atc(2-), [( et hy 1 cnodi n i t rolo )t e traacet at o ]-, dlsodlum
Calcium EDTA
Calcium cthylprvediBminctetraacetatc
01—sodium calcium CDTA
EdatKeniil calcium disodium UD
CaC,0Hi,.2Nd
Antal i n I1F.RCK
Calci atc( 2- ), f ( c t hy] f-ncd I nl t r i 1 o )te tra ace t oto ]-, dfsodium
Calcium diaodiuin cdctate USP,CTCP
Calcium disodium etliy lencd i ani nc tP t raacet a t e USP
Calcium disodiuni ( et ny lcn«d i iti tr 11 o )lc t raacctatc MERCK,USP, CTCP, FCC
Calcium Disodium Vcrsonnte HEI1CK
Edothamil calcium d i 3v>d i urn MERCK
Etdylcncdiaminetetraacctic acid, calcium disodium chclate MERCK
Mosat i 1 KF.KCK
CloHlt.20!12.Ca.2Na
Calcium disodium edetalu dihydrate FCC
Calcium disodium EDTA dihydrate FCC
Calcium disodium et hy 1 <-ncd 1 am I ne t ct r aacct a t e dlhydrntc FCC
Calcium disodium ( e Itiy li:ncd i nl t r I I o )t r-t raacet ale dihydrate FCC
Sodium [(<>thylenrd!nltrilo)tc!traae<-t«l<-}calclatct >l/\,[ Ca(CloMi7N ,(),)]
, d Iliy dra tp
377
-------
Nz08CaC, DH, , ,'J.IU. xOHj
ftntallln CDF
Calclat<>(2- ), [ ( et hyt cnedlnt tr 1 1 o)te t roaeet ato ]-, dlsodium, hydrate
Calcium tit-iodlum edi>tatAN,USftN-A
Calcium dlsodlum (et hy Jpnedi r\I t ri 1 Q )tc t raacetate hydrate USftN
Calcium diaodiura vorsenate USAN
li USAN
ium ^inc rmA Il'CMTN
5cc|u<;c P t hylfpfd I ami nc te tr ooc<-t ic acid
Zlncnl <.'(;'-), I ( i-1 li»/li'i\i d I ni t ri 1 <. )tc t rauct-toto ]~, dlaodiui
1
Acetir nci.l, ( c-t hy I <-m-d I n i t r i lo )t t- 1 p a- , iron salt
UlhyJro.H-n fcrroui f.DTft IECMTN
KOTA tron( I I )
S.-qu^fltri-n.- H.'.Tt HTHTN
, ,,, b
Act'tlc ncl.l, { • t livlv-iidU nl t rl lo) tetra-i Iron complex, sodium salt
!'tlt« iron -niiHu'i!
37'd
-------
Graph I
20
19
18
17-
16"
15
m
13
12
11
10
c
£8
£
to
w
lu
2 - -,.-
100
.: .I:
U
In • \
Production of Na;.EDTA
m
SSgr
_|._
i
, 0
J4
•ILf-IL
iUr*:l
'jo
6
Trf r
: - --rfn ' I
i^Eth-T^
•s:^-.-
j
u-i
Per Cent of Production Sol
& "'/>
ci)
^o' '61 '62
(•> i
•6Y '6IJ
G
-------
_880
j 660
I i
^°J
220
Production of EDTA and Its Salts
-—r~T~r
0 ..
160
; i"
i 1201
i
A0'
i)"
TEH
28oi L
b:
w
rr|-
-1
NaFeEDTA
l~
-
260
g t -L:_!
° 2UO!
o
•H
.
220! :
, 200 j
i-^^rr
360 i
320
280.
200
...:(:-
-r:-1-
Na-jEDTA
i 2800
2100 :
p-T—p
I ! !
1UOO I
700
G
Na2EDTA
"0
(£
^
'55 '56'
1 I EDTA
•'57' '''58 '^59 '60*"'61J '62 '63
15' -''66 ''6? '60'•''<:
70 -71
380
-------
A number of the formulas given are incorrect in that they retain the
H's displaced by the metal ions.
II. PRODUCTION
Production figures for the tetrasodium salt of EDTA are available
for the years 1955-1971 and are presented in Graph I; this salt is the
material from which EDTA itself and the other salts are prepared. Avail-
able statistics for EDTA and other salts are shown in Graph II. None 01
these figures should be taken literally because it is only the tetra-
sodium salt which is being "made." It, at least, shows a clear upward
trend, seemingly at a geometric rather than the earlier algebraic in-
crease; the percent actually sold is moderately steady at about 70.
Prior to 1968 about 50-55% of EDTA production was sold, but then the
figure dropped sharply to about 30%. For the NaFeEDTA, there was a
50-50 chance of reported sales exceeding reported production. The 1971
U.S. Tariff Commission Report indicated the following companies to be
producers of the noted EDTA's: Ciba-Geigy Corp.—Naif, Nas-, Na2~,
EDTA, Kit, NaFe-, Mn-, Na2Cu-, Na2Ca-, Na2Zn-; Crest Chemical Corp.— Naif;
Dan River, Inc.— Na^-; Dow Chemical Co.— Na^-, Na2 , EDTA, (NHit)tf,
(NHi+)2-, Na2Ca-, Na2Zn-; Eastman Kodak Co.—Na2-; W.R. Grace & Co.—Naif,
Nas-, Naa-, EDTA, KI+-, NaFe-, Mn-, Na2Cu-, Na2Zn-; Hart Products Corp.—
Naif-; Millmaster Onyx Corp.—Naif.
III. USES
The following table (III) was presented in Chemical Economics
Handbook (1967), but it was not exclusive to EDTA.
-------
CHEMICAL ECONOMICS HANDBOOK, Stanford
Research Institute, Menlo Park, Cali-
fornia, p. 512.5020R
Table III. Estimated Markets for Aminopolycarboxylic
Acid Chelating Agents, 1965
Textiles 30%
Soap and Cleaning Compounds 20
Water Treatment 15
Miscellaneous Chemical Processing 15
Agriculture 5
Rubber Processing 5
Metal Cleaning and Electroplating 5
All Other (Largely Pulp and Paper Processing <10
Source: CEII estimate based on communication with industry
CEH elaborated on these uses as follows: textiles - improvement
of dyeing evenness, extension of life of alkaline bleaches, water
softener in cleaning operations; soap and cleaning compounds - water
softener, foam stabilizer, builder; water treatment - prevention of
scale in boiler water, scavenging of limey deposits; miscellaneous
chemical - improvement in product quality and yield, catalyst recovery
in petroleum products, mineral flotation separation adjunct, rare earth
separation, pre-ion exchange treatment; agriculture - correction of
mineral deficiencies, water softener for spraying operations; rubber -
copolymerization activator, metal scavenger; metal cleaning - "rust"
and lime remover, metal scavenger, etchant. Water treatment was the
anticipated area of fastest growth.
EDTA has some important, but probably only small quantity, use in
analytical chemistry as a titrant for metals.
-------
Considerable amounts (relative to human contact) may be used in
foodstuffs, the disodium and the calcium disodium EDTA being the only
salts allowed. Table IV, adapted from the 1972 edition of the Handbook
of Food Additives, lists the allowable amounts and the specific foods
permitted to contain them; there have been no changes in the allowable
amounts since 1960,
Table TV. Regulatory Status of Direct Food Additives
CaNa?EDTA
121.1017
Material
FDA Regulation
Limitations
Na2EDTA
121.1056
Alone, as food additive
33 ppm max in canned
carbonated soft dunks
110 ppm max ID canned
wlnte potatoes
310 ppm max in canned
cooked clams
275 ppm max in canned
cooked crabme.u
2a ppm max in du-tilled
alcoholic beverafit'^
75 ppm max in non-
standaidi/ed dit -- in^o
3)0 ppm max in canned.
eookt'd, dried bma bean*
25 ppm max in fomienletl
m'llt boveiages
75 ppm iiijix in French
r(r) ppiu max in mayon-
200 ppm max in canned,
7T> ppm max in oluo-
r:i.n;;,irino
1(K) pptn max in pf c in pie
filliri}:
ii'-il) ppm max in pickled
tabbane
^1*0 ppm ma\ m pukled
ruiiinibfi s
1(H) ppm in.ix in potato
i-il.id
1. Alono, as-food additive
150 ppm max in aqui-ou-
mullivitamm prepara-
tions, with iron salts sis
stnbi U?.er for vitamin 15,,
145 ppm max in canned.
black eyed peas
165 ppm max in canned
cooked chick peas
Mil ppm max in canned
kidney beans
000 ppm max in canned
sliawbefiy pie fi!lin>:
3i"5 ppm mnx in coaled
sausage
75 ppm max in non-
btandardizi-d dressini:
315 ppm niaj. in din d
b:in.inti romponont cil'
ready-local cereal pmd-
nets
75 PDIII max in I'loueh
tlrt^sin;;
HK) ppiu m,»x in fin/en
whil;' pol.ilocs, nicltidin,"
cut ikitaKH";
Wl |ipin in.ix in i;efiltf
fu-h balU or palde-., m
cludini; liquid juiUifi;
tnt'dium, to iidubit di -•
C(>k»iatn>n
7fi ppm max in masim
IHlise
-------
800 ppm max- in proc-
essed, dry pinto beans
75 ppm max in salad
dressings
100 ppm rji.ix in sandwich
sprv.uK
7"> pi'pt max in .vmces
2."0 ppm in, IN MI canned,
75 pprn max in salad
dressing
100 pprn max m snndw ich
spread
75 ppm max in sauces
2. In combination with cal
cium disoduim 1COTA as
food additive:
75 ppm mnx in non
standardized dressing
75 ppm max in French
dressing
75 ppm max in mayon-
naise
1000 ppm max (dry-wo'C^1
basis) in noninitrilivi'
sweeteners
60 PJ .11 n.,ix in spicv ex-
tractives in M'luble car-
riers
ICO ppm nv,>\ in :atificial-
!y flavored 1 mon and
oran;'i spiead-.
In combination with di-
Fodiiun Kl 1 1 A u ^ kuni addi
tive:
7,') ppm ma\ in non
stanclardi.'ul di e-: in^s
75 ])p'ii irux in Flench
dress-inu
l~j ppm max in m;i>on-
n;nse
75 ppm max in salad
dressing
100 ppm max in sandwich
spread
75 ppm max in sauces
Pioduct sprcihcations ap-
ply
T.E. Furia, in Chapter 6 of this Handbook, discussed the purposes
of using EDTA's in various foods. Fats and oils, and foods containing
them, were protected by a synergistic combination of EDTA and antioxi-
dant (BHA, BHT, ascorbic acid, etc.)- Aqueous vitamin preparations,
especially vitamin C, or oil soluble vitamins such as A, D, K, and K
were stabilized by EDTA's (in conjunction with antioxidants for the
oil solubles). Processed fruits and vegetables suffered less color
changes and alterations in flavor or texture. Fish and shellfish had
improved color stability and less tendency to form the glass-like crys-
tals called struvite. Wine, cider, and vinegar showed much less ten-
dency to form precipitates. Milk was kept from developing off flavors
resultant from copper contamination. Various benefits accrued to the
beer and sausage manufacturing processes.
-------
IV. CURRENT PRACTICE
No information concerning handling and transportation regulations
or disposal methods was found.
V. ENVIRONMENTAL CONTAMINATION
No information concerning environmental occurrence was found.
VI. MONITORING AND ANALYSIS
A variety of chromatographic, spectrophotometric, and titrimetric
procedures has been developed which bypass the presence of the usual
cations associated with EDTA systems; the cations may be detected and
quantitized by standard methods in inorganic analysis.
Heinerth (1968) detected EDTA in detergents by thin layer chroma-
tography after preliminary extraction and removal of interfering salts.
The medium was Kieselgel G.
Yamagata et al (1969) developed a thin layer chromatographic
technique especially useful for separating the EDTA used in foods from
amino acids, particularly aspartic. Their medium was the cellulose pow-
der Avicel SF, and the solvent system n-butanol/acetic acid/water (1/2/2
by volume). The spot was detected by spraying with acetic acid, cobal-
tous chloride, and hydrogen peroxide. The Rf for EDTA was about 40%
greater than that for aspartic acid. The limit of detection was 1.5 yg.
Mihara et al (1970) analyzed food for EDTA by gas liquid chroma-
tography after conversion to the methyl ester by simply refluxing in
acidic methanol (claiming that diazomethane or boron trifluoride-
methanol esterification was unsuitable). Detection limits were 8.4 ng
and 12 ng on 4-mm X 1.08-m 5% QF-1 on Gas-Chrom Q at 175° or 3-mm X
1.5-m OV-1 on Gas-Chrom Q at 185° columns, respectively.
.385
-------
Rudling (1972) analyzed for EDTA in water or sewage in the presence
of nitrilotriacetic acid (NTA) and diethylene-triaminepentaacetic acid
(DTPA) by conversion to the methyl ester and gas-liquid chromatography.
The esterifying agent was boron trifluoride in aqueous methanol. The
chromatographic system consisted of a 100 X 0.2 cm i.d. glass column
packed with 5% (w/w) OV-17 on 100/120 mesh Aeropak, helium carrier gas,
and a flame ionization detector. The EDTA ester eluted at 12 minutes
into a 10°C/min. programmed rise from 150°C. The minimal concentration
detectable was 10 yg/1 (about 10 ppb). A solution containing 0.2 mg/1
of EDTA was analyzed without interference from 2 mg/1 concentrations of
Cd(II), Cu(II), Fe(III), Ni(II), or Zn(II).
Menis et al (1956) measured EDTA by forming a Cu(II) complex and
measuring the absorbance at 250 nm. Good results were obtained at con-
centrations down to 0.1 g/1, not quite as good in the 0.025-0.1 g/1
range. At the 0.05 g/1 level of EDTA, interferences came from Cr(VI),
Ni(II) , and Co(II) (the latter only when present in amounts over 10% of
the EDTA).
Vogel and Deshusses (1962) determined EDTA in wine by forming a
complex with Co(II), then oxidizing with peroxide to the Co(III) complex,
and measuring the absorbance at 530 nm. A minimum of 2 ppm EDTA was
detected.
Stahlavska and Malat (1965 and 1965) analyzed pharmaceuticals for
EDTA by using it to displace various heavy metals from phenolic chelates,
and measuring the remaining absorbance. Concentrations of EDTA as low
as 0.7 yg/ml were detectable.
Suk and Smetanova (1965) added excess Bi(III) to an EDTA solution,
adjusted the pH to 2.0, added bromopyrogallol red, and measured the
y'.t.
-------
absorbance of the Bi-catechol complex at 635 nm.
Mottola and Freiser (1967) demonstrated the feasibility of measuring
sub-micromolar quantities of EDTA by the inhibiting effect it has on the
catalysis by Mn(II) of the oxidation of malachite green by periodate.
However, many of the non-alkaline earth metals and also other polyacetic
acid complexants interfered severely.
Kross (1968) patented a method for determining EDTA in meat products.
The EDTA was complexed with Ni(II), the complex destroyed by oxidation,
and the liberated Ni(II) complexed with dimethylglyoxime for spectro-
photometric measurement at 430 nm.
Ishihara (1968) added excess acidic zirconium solution to an EDTA
sample, then added xylenol orange and measured the absorbance of its
complex with the non-chelated Zr at 530 nm. Various common cations and
anions interfered, otherwise the minimum detectable amount of EDTA being
50 ug.
Shimokawa and Horibe (1968) determined EDTA in food by measuring
the absorbance of the cobalt complex after removing interfering amino
acids by passing the dissolved sample over a column of the anion-exchange
resin Amberlite IR-45 at pH 2.1. The limit of detection was 0.4 mg.
Saito et al (1968) used the cobalt/peroxide method (Vogel and
Deshusses, above) for EDTA in sake. They reported a useful range of
5-600 ppm, and cautioned that the pH of the final solution must be 3.0
to prevent interference from any amino acids. Common inorganic and
organic acids and salts at concentrations below 0.1% did not interfere.
Bruno et al (1969) used this same method for EDTA in fruit juice
but measured the absorbance at the slightly higher wavelength of 535 nm.
Their errors on spiked samples were ± 5%.
387
-------
Bhattacharyya and Kundu (1971) determined concentrations of EDTA
in the yM region by adding excess Fe(III) and measuring the absorbance
of the non-chelated and chelated iron at 305 and 258 nm, respectively.
Most common cations did not interfere.
Krowczyriski and Banaszek (1958) measured EDTA, Na2EDTA, Na2CaEDTA,
and Ca2EDTA in pharmaceutical preparations by titration with Fe(III)
using sulfosalicylic acid indicator.
Hennart and Merlin (1958) checked on the purity of Na2CaEDTA by
separately determining the Ca and Ca plus Na. A sample was ignited to
the mixed carbonates and divided in two. One portion was dissolved in
acid, adjusted to pHIO, and then titrated with EDTA to determine Ca.
The other portion was dissolved in perchloric acid/propanoic acid and
the excess perchloric titrated with pyridine/propanoic acid with mala-
chite green to determine Ca plus Na.
Clinckemaille (1968) determined EDTA in detergents by titration
with Cu(II) and the indicator commonly written as PAN; one percent of
any nitrilotriacetic acid present would titrate under the conditions
used and would have to be determined separately for accurate work.
Heinerth (1968) also used Cu(II) to titrate EDTA in detergents,
at pH 4 and 60°C, but with the indicator polyacrylonitrile; under these
conditions hydroxy-EDTA also titrated.
Huber and Tallant (1968) titrated EDTA solutions with Pb(II) using
constant current potentiometry as an endpoint indicator. Concentrations
at the sub-mM level were determined with good accuracy. The common
anions and Ca(II) did not interfere; phosphate ions at the mM level
interfered with the normal way of running the analysis but could be
counteracted by plotting the experimental data; no Mn(II) could be toler-
ated.
388
-------
Treffler (1968) discussed an extraction/titration technique for
determining EDTA in powdered alkaline cleaning compositions.
Vanderdeelen and Van den Hende (1968) added excess bismuth ion to
an EDTA solution, and then titrated the uncomplexed Bi with standardized
EDTA using pyrocatechol violet indicator. Under the conditions used,
only Hg(II) and oxalate ions interfered.
Blijenberg and Leijnse (1969) titrated EDTA in blood or urine with
Cu(II) using the indicator pyridylazonaphthol and a visual or colori-
metric endpoint. Large excesses of Ca(II), Mg(II), phosphate, or ci-
trate were non-interferants.
Groninger and Brandt (1969) determined EDTA in fish and shellfish
by titration with Th(IV).
Reuge (1971) determined CaEDTA in protein solutions by titrating
with Zn(II) after precipitating the Ca with oxalate.
Milwidsky (1971) reported a method for determining EDTA in the
presence of detergent phosphates. A sample containing at least 0.1 g
EDTA was adjusted to pH 2.5, Zn(II) added, and the pH readjusted po-
tentiometrically with NaOH to 2.5. The amount of NaOH corresponded to
the amount of EDTA. Any nitrilotriacetic acid present interfered.
Titrimetric procedures for determining the purity of calcium di-
sodium EDTA and disodium EDTA were described in Food Chemicals Codex
(1972), pages 128 and 259, respectively. The former was titrated with
thorium against xylenol orange. The disodium was converted to the
calcium complex, then titrated with NaOH against hydroxy-naphthol blue.
VII. CHEMICAL REACTIVITY
A. Environmental and use associated reactions
Most of the reactions EDTA and its salts undergo are simple
389
-------
complexations of polyvalent metal ions after displacing sodium atoms or
another metal from the EDTA. In general the complexes are formed more
readily at high pH than at low because the H's are more apt to be
ionized at the former; preformed complexes of tri- and tetravalent metals
are stable at pH's ^ 1.
Deleted because of copyright clearance
Cheronis and Schatz (1958) claimed that EDTA catalytically degraded
basalt, limestone, or shale rocks. They showed that an aqueous EDTA
solution covering the rocks gradually increased in pH and acquired color-
ation, whereas omitting the EDTA resulted in no such changes.
B. Aspects with biological implications
Hashimoto (1966) demonstrated that EDTA interfered with the ability
390
-------
of volcanic ash or red soils to fix phosphorus, but enhanced this ability
in a calcareous whitish soil.
Ikehata et al (1967) studied the effect of EDTA on the formation
of flocculated A1(OH)3 in water treatment processes.
Anghileri (1968) demonstrated that EDTA could bind to serum
albumin and possibly interfere with the albumin's metal-binding ability.
Singh (1971) thought that some light on the toxic side-reactions
from therapeutic EDTA dosing might have been shed by showing that, in a
non-chelating fashion, EDTA could dissociate both ionically and non-
ionically linked complexes of polymerized DNA.
VIII. BIOLOGY
A. Metabolism
1. Absorption
In a study on normal human adult males Foreman and Trujillo (1954)
found that only about five percent of an oral dose of CaNa2EDTA was
absorbed, and that almost none of a skin applied dose was absorbed.
Wallace et al (1955) discussed the absorption from soil by various plants
(see VIII.3 and 4, and X. D. for other findings of this study).
Foreman (1959) reported that rats didn't absorb much EDTA from an oral
dose, but did absorb more if they had been fasted beforehand. Spencer
(1960) studied the absorption of oral doses of CaEDTA or N32EDTA in
man at the rate of 6 gm/day for 6 days. Some of the subjects absorbed
a little of the dose, others none at all. Wallace and Mueller (1966)
applied FeEDTA (both Fe and C isotope labelled) to an alga, but could
not determine if only the Fe was absorbed, or the whole complex
absorbed and EDTA portion immediately eliminated. Kealy et al (1969)
indicated that chicks fed Na4EDTA absorbed more than half.
-------
Wynn et al (1970) fed male rats diets containing up to 10% by
weight of NaaEDTA for 13 weeks. Some of the EDTA was absorbed,
apparently as the CaEDTA form, but never exceeded a serum level of
1 mg/100 ml.
2. Excretion
Foreman and Trujillo (1954) subjected normal adult male humans to
intravenous, intramuscular, oral, or skin doses of CaNa2EDTA at levels
of 2002.2 mg, 1002.2 mg, 1.5 mg, and 1002 mg, respectively. Within the
accuracy of their analytical method all of the i.v. and i.m. dose was
recovered in the urine in a 24 hour period. Of the oral dose a minimum
of 91% was recovered within three days in the feces and urine at a 23/1
ratio. At most 0.001% of the skin dose appeared in the urine. The half
times for blood clearance after i.v. or i.m. dosing were 65 and 90
minutes, respectively; there was no detectable EDTA in the blood after
oral or skin dosing. There was no indication of the presence of meta-
bolites in the urine. The renal clearance value of 680 ml/min. after
i.v. dosing indicated that glomerular filtration and tubular excretion
both played parts In the clearance.
To study the stability of EDTA in plants Wallace et al (1955)
grew orange seedlings for 60 and 110 day periods in soil containing EDTA
bearing isotopically labelled nitrogen, then water-extracted the leaves
and chromatographed the extract over cation and anion exchange resins.
EDTA itself is not retained on cation resins, but a considerable fraction
of the radioactivity was found on the cation resin after both periods of
growth, indicative of degradation of the EDTA.
Foreman (1959) reported that rats given EDTA parenterally excreted
97.5% within six hours, with a blood turnover time of 57 minutes after
392
-------
i.v. dosing. Clearance from the blood occurred only through the kidney.
The remaining 2.5% was released slowly, possibly having been bound to
iron strongly fixed to something. Tubular secretion and glomerular
filtration were involved. These results paralleled the author's work
with humans (above).
Spencer (1960) reported that essentially all of an EDTA dose given
to three human subjects was eliminated in the urine in a 24 hour period,
in agreement with the Foreman study (above), but, perhaps, experimentally
more valid.
Darwish and Kratzer gave 7.4 ym doses of C-14 labelled EDTA orally
to laying hens which had been colostomized. The serum plasma EDTA level
peaked at about 0.1% of the dose at about one hour, then dropped rapidly
and leveled to about 1/4 this level, where it remained for almost two
days. Carbon-14 in the respired air peaked at 7 and 28 hours in one
bird, at 7 and ? hours at a higher level in the other bird (experiment
with this bird terminated at 42 hours); there was still activity in the
first bird at 110 hours. Urinary C-14 peaked at about 11 hours in the
42-hour bird, about 1/3 as uric acid. After 144 hours recovery of the
dose from one bird amounted to 4% in the expired air, 9% in the urine,
52% in the feces, and 1% in a G.I. tract washing.
Havlicek et al (1968) gave adult rats intraperitoneal injections
of EDTA (Ca and Y cations, 1 and 100 yM doses, neither being a factor
in the results) labeled with C-14 in the -C*02H, -C*H2C02H, -C*H2N-
(CH2C02H)2 positions. After 24 hours about 1.2% of the C-14 in the
first two of these, but only 0.05% of the C-14 in the last, showed up
in the expired air. In the same period 95 ± 6% of the dose showed up
in the urine, and about 1/4% in the feces (C-14 in the C*02H). There
393
-------
was some evidence for most of the decomposition occurring in the
kidneys.
3. Transport
Foreman and Trujillo (1954) determined that one hour after an i.v.
dose of CaNa2EDTA in a male human adult the level of EDTA in the spinal
fluid was only 1/20 that in the blood plasma, indicative of very slow
transport across the blood-spinal fluid barrier.
Wallace et al (1955) grew bean plants in nutrient solutions con-
taining varying amounts of Na2EDTA. Table VI indicates that there
wasn't any linear relationship between the concentrations of EDTA in
the nutrient, roots, or plant top. The plants seemed to be able to
absorb the EDTA faster than they could transport it.
Tanton and Crowdy (1971) reviewed the use of PbEDTA as an agent
for the study of transport in plants.
TAMI. 1C VI-Yii i n. HI) I A \M> MIM mi ("IIM-I.M- !«• Pi u Ci sr I>K Dm \Vi u.m nf Hi \v
I'l \N ts I". HOW \ II I> \1S IN \! I l<] 1 \ I Sill I I I0\>. CON I \ININ«. VAKIOI S C.I l>tl I N 1 h \ •
1 IONS 01 N \:IC1) l'.-\ lit I \\ M II CONST \N I 1.1 \ I I s. ol Mu ItoNt'1 utl.Ms.
EDTA in
N.i-EDTA . VV.iliT M.lul.lc cotnpir.il.lc
in inn u-iit Dry ICI) IA nlru^
volution \M-i.ht in pi ml* Fe Mn Zn Ic.ivi^*
()>|'in) d'/plnu) t',1 (ppm> (ppm) (ppin) ('; )
Top, Root* Tf>|)> RO..IJ Tops Roots Tops Ronti
0 c!O T T I.I IO1.1 10 SI 7't .Ml T
Him
_'OUI
M-1 :i'i Hi:
J'l.S 0 (HI
I.. I 011.1
111 DON
s rt7 "Hi
-"i i i 11 (iiv.i
ii-' r..> n.ii
T o ID
III II IJ Hit i
IJ 01.' 111
I I II Jii 7n
I'l I I)'. 1 (7
iii -'/'n -'in
P 4,1,, II*'" 17 .'*• 7 i," :i'l •.•!••" L'I. 7(1
i.--IMO-.I i '' _ "ii ii'.'. __^>_s !'''-'„ ^_hi :"_ 'J ''"'
\S.)!i.c p'.int niittii.iK . ,\L- !>o-iti\c tc^t uit.i rc-.i.'cnls even though ICDTA ^ nut prr*Li'.t.
k. Distribution
Foreman and Trujillo (1954) calculated that shortly after i.v.
injection, EDTA left the blood stream and permeated the body's entire
water supply, exclusive of spinal fluid and red blood cells.
Wallace et al (1955) measured the distribution ot N-15 labelled
EDTA in orange cuttings; their results are given in Table VII.
-------
u-.ii X Cmiix-i. HKOM EDTA. C\H.VLVTI.I> IKON E««'II-.\I.KSIV ANI>
HAM.U C> IIIMJI Si'i-riii.n K''-L»ni-i.LD X.iFi-EUTA n>» Kf
Plant part Lalxiletl >
plai
(Vf
HI
1 It
It
l-'I
r.
ti plants
n> Cpi"»)
81
SS
10-'
(ij
41
113
diiicivncc
(ppin)
L'8
21
31
~>",
IS
57
treated jil.-ut
(pprn)
37.1.
.">-'. 1
5on
s.'.s
Lffdf mnrcins O.O018S
Inner |',ir t «Jt'leaf. ... (Mfff,1'..1
l.i-at \vin »»IX)t-'J
K'tiulca . (IDlllllU
Dark . 0(101'.I
Wo,..!. ...... , (Hums
Callus IUIIH1K
Kuitlntk. ,. . ()«()•. I«t HI) I i'.S 8'.8
Root I" 8
F'lr.o ri>',l^ .
lll.O
. . .
'•Kr,,m tl-f l.,1,d, ii \ i.n.tiiU Tin-l.il»-1«l .\ ».«» .lonvc.l fiwn EDTA.
Foreman (1959) reported that no organ of the rat concentrated
CaNa2EDTA to any extent. The EDTA was distributed over a volume a bit
greater than the extracellular space.
Matsuda (1968) confirmed, with tomato plants, Wallace's observation
that EDTA concentration was greater in roots than plant tops.
Weber (1969) gave adult rats i.v. injections of C-14 labelled
CaNa2lDTA and examined certain organs autoradiograpMcally 24 hours
later. The kidneys showed accumulation in the proximal tubules but
not in the glomeruli; the duodenum showed activity in the mucosa and
crypts. Lesser activity was evidenced in the liver parenchyma, bile
ducts, and blood vessels. The pancreas and adrenals showed no
accumulation.
Plagne et at (1969) injected C-14 labelled HgEDTA into rats and
found accumulation only in the renal cortex.
Tanno et at (1972) gave rabbits i.v. dosage of In*EDTA and mea-
sured the disappearance with time from the blood and organs. After 30
minutes the accumulation was (in decreasing order): kidney, blood,
lung, pancreas, liver, marrow, spleen, brain. All decreased at about
the same rate except the pancreas which increased up to 100 minutes be-
fore dropping.
3?5
-------
B. Physiological Effects
Kabakow and Brothers (1958) gave a number of adult human subjects
an i.v. injection over a four-hour period of 4 gm Na2EDTA in 250 cc of
5% aqueous dextrose. On average the serum Ca was depressed 1.9 mg%.
About half the time minor hypotension resulted, at most 15 mm systolic,
10 mm diastolic. Throughout the infusion period a tolerable, burning
pain was felt from the point of insertion and downstream. Accompanying
this in 1/3 of the subjects was a sensation of prickling around the
mouth and warmth elsewhere on the face.
Vozar and Bobek (1958) gave oral doses of Na2EDTA to guinea pigs
and rats, resulting in strong decrease of y-globulin. Zizine (1958)
reported that feeding rats CaNa2EDTA as 1/2% of their diet significantly
reduced the thyroid activity. Fujita and Imai (1958) gave rats 10 daily
injections of 50 mg/kg of CaNa2EDTA, or 11 doses of 5 mg/100 gm of
Na2EDTA. Results of the Ca treatment were: decrease of HIOif-Schiff
positive substances in the heart, regressive degeneration of the liver,
degeneration of the kidney, slight congestion with many large nuclear
cells in the spleen, and hemorrhage with wide cells in fhe lung. These
results were repeated after the other treatment but to a lesser extent;
in addition the vascular and lymphatic systems and capillaries showed
lesions and bleeding. Sacca et al (1958) thought that the side effect,
osmotic nephrosis, from treatment of Pb poisoning with CaNa2EDTA derived
from the Na atoms.
Vozar (1959) studied the effect of Na2EDTA on Cu in rats. Feeding
40-80 mg/100 g/d for three days produced a marked decrease of hepatic,
renal and skeletal musculature Cu content. Running the experiment for
20-40 days resulted in deposition of Cu in skeletal muscle and cerebral
396
-------
cortex gray matter concurrently with removal from liver, kidneys and
heart.
Kelenyi and Kasza (1959) gave rats a 60 mg dose of Na2EDTA by interscapu-
lar injection, resulting in the development of a tumor-like edema at the
injection site, attributed to the binding of Ca.
Vozar and Simko (1959) examined the blood of rats dosed with 40
mg/100 g/d of Na2EDTA for 3-7 weeks. After three weeks there was no
change in the number of segmented neutrophile leucocytes or erythrocytes,
but the latter's hemoglobin content had decreased. The number of lympho-
cytes and the total leucocyte count also decreased. The blood gradually
returned to normal during the 4th-7th weeks of treatment.
Foreman (1959) reported that EDTA parenterally administered to rats
caused hydropic degeneration of renal proximal tubules, reversible on
cessation of dosing.
Spencer (1960) reported that i.v. dosage of humans with 4 g Na2EDTA
over a four hour period produced in the urine only about 65% of the
extra Ca which the EDTA was capable of complexing, without affecting the
serum Ca level. A similar test with CaEDTA produced only 81% of the ex-
pected excess in the one day test period.
Sullivan (1960) reported that a diet containing 4% MnEDTA caused a
reversible, severe iron deficiency anemia in immature, but not in adult,
rats.
Smith and Kerby (1960) gave rabbits a number of subcutaneous injec-
tions of Na2~ or CaNa2EDTA resulting in urinary excretion of acid muco-
polysaccharides of brief and widely variable duration and extent.
Albach (1961) reported that the effect of Na2EDTA on serum Mg level
on human males was a function of age. Below 25 years no effect was
397
-------
observed; above, there was an average drop of 37% in two hours after
injection. Normal levels recovered within 12 hours.
Remagen et at (1961) gave daily injections of 150-200 mg/Kg EDTA
to young rats and rabbits for 14 days. Results included significant
lowering of serum Ca and serum alkaline phosphatase, considerable Ca
excretion, and change of blood pH to an alkaline condition.
Oser et at (1963) reported that feeding rats for two years and dogs
for one year on a diet containing up to 250 mg/Kg of body weight had no
effects on physiological responses or mineral metabolism.
Daniel and Erwin (1965) reported that Na^EDTA had a stronger effect
than Na2EDTA on the contraction and relaxation of rat uterus which
depend on Ca and Mg ions.
Schane (1965) gave spayed rats i.p. doses of 0.6 mmole/Kg EDTA and
found that within one hour there was an increase in uterine phosphorylase
activity similar to that seen 48 hours after estradiol treatment. Similar
treatment of cows, guinea pigs, mice, and rabbits gave intraspecies in-
consistency.
Neu et al (1966) demonstrated that the combination of EDTA and Tris-
HCl effected the release of acid-soluble nucleotide material from E. coli
in 6-10 minutes. The viability of the cells was unchanged.
Hamilton-Mi Her (1966) demonstrated that Na3EDTA and Na^EDTA, but
not CaNa2EDTA or MgEDTA, greatly increased the outer membrane permeabil-
ity of various common bacteria at concentrations as low as 0.1 mM, with-
out impairing the viability. Nucleic acid material leaked from the cells.
The maximum effect was found to occur at pH 7.4-7.6, coincidentally, per-
haps, the optimum pH for chelation of Ca by EDTA.
Watras et al (1966) gave rabbits i.v. doses of 20 mg/Kg of CaNaaEDTA
398
-------
on alternate days for 48 days. There was a significant reduction in the
amount of Fe stored in the liver, spleen, and long bone marrow.
Rasmussen and Cooper (1968) and Cooper et al (1968) reported that
ethylene production in the calamondin (citrus) tree was stimulated by
CuEDTA, but not by FeEDTA.
Schwiegel (1969) found that the permeability of the main capillaries
of rabbits and rats to the dye Evans blue was increased not at all by
CaNaaEDTA, somewhat by MgNa2EDTA, more so by NaaEDTA.
Dubina et al (1969) reported that seven consecutive daily i.p. in-
jections of 70 mg/Kg of EDTA to adult rats temporarily decreased the
liver mitochondria activity but did not affect the activity of the res-
piratory enzymes dependent upon Cu or Fe. Different results had been
found in an in vitro study.
Fiedler and Hartmann starved guinea pigs for 16 hours, then gave
them s.c. injections of 0.3 mmole/Kg EDTA, 0.6 mmole/Kg MgEDTA, 0.15
mmole/Kg ZnEDTA, 0.6 mmole/Kg ZnEDTA, 0.03 mmole/Kg Zn2EDTA. They then
measured serum glucose at 1/2, 1, 1 1/2, 2, and 3 hour intervals. EDTA
itself elevated the glucose level by 29% at 1/2 hour, and prevented re-
turn to normalcy by 2 hours. Co-injection of an equivalent amount of
alloxan (non-hyperglycemic and non-diabetogenic in guinea pigs) caused
the glucose to rise 50%, and remain up 30% at 3 hours. The effect of
the Mg and Zn EDTA's was less marked. It was concluded that the EDTA
acted by epinephrin secretion stimulation, and not by Zn complexation.
Lie and Brotonegoro (1969) found that FeEDTA, and K2EDTA to a
lesser extent, interfered with nodule formation on the roots of pea
plants.
Wynn (1970) fed rats varying amounts of Na2EDTA for three months,
399
-------
with the effects on food consumption and weight gain in Tables VIII and
IX, respectively.
Table VIII. Effect of EDTA on Average Weekly Food
Consumption in Adult Male Albino Rats
Diet consumed (g)
Na.H.EDTA
End of
Control
1.0% 5.0% 10,0%
1
2
3
4
5
6
7
8
9
10
11
12
13
341
164
169
'174
168
152
175
165
166
163
165
151
145
141
171
158
162
193
147
162
178
177
174
182
144
137
111
137
129
108
115
122
134
145
148
157
158
135
144
116
98
85
76
61
86
69
78
85
80
71
84
53
Table IX, Effect of EDTA on Average Weekly Body
Weights of Adult Male Albino Rats
Body weight (g)
Na2HjEDTA
I^UU Ml
week
0
1
. 2
3
4
5
6
7 .
8
9
10
11
12
13
Control
116
171
223
273
313
334
351
373
390
406
422
431
437
442
1.0%
121
171
218
255
286
310
320
335
36!
380
403
399
409
418
5.0%
120
130
156
177
185
188
203
209
235
248
267
261
276
305
1 0.0%
123
116
119
119
124
115
115
119
122
129
145
146
136
127
In both tables differences in only the 5 and 10% columns have statistical
significance. Animals on the two higher concentrations developed diarrhea
by the third day on the diet and were subject to it throughout the experi-
ment, consuming twice as much water as the controls. Some of the animals
1*00
-------
were subject to priapism (all of the 10% and one-fifth of the 5%) on-
setting in the Initial four weeks. There were no hematological differ-
ences at the end of the test. Gross and histopathological examination
of the internal organs showed no abnormalities, other than pale livers
in the 10% group.
Vohra and Bond (1970) fed fowl diets ranging 0.5-4.0% in Na2EDTA'2H20.
The high levels depressed weight gain, hematocrit levels, Fe levels in
the blood, liver, and kidney, and Zn level in bones; renal Zn was in-
creased.
Dvorak (1970) found that increased presence of K and Mg ions in
the urine of rats dosed i.p. with Ca- or ZnNa2EDTA was solely a function
of the Na ions in the dose.
Fritz et al (1971) fed chicks diets containing various amounts of
EDTA, Na2EDTA, and CaNa2EDTA to study the utilization of dietarily
marginal amounts of Ca, Fe, and Mn. Amounts up to 1600 ppm had no
effect on the Ca. Incidence and severity of perosis from 1600 ppm of
Na2- or CaNa2EDTA or 800 ppm of EDTA were equivalent. The same amount
of EDTA depressed growth and hemoglobin. Depression of hemoglobin and
hematocrit also resulted from 800 ppm of the two EDTA salts.
IX. ENVIRONMENTAL EFFECTS
A. Persistence and/or degradation
Hill-Cottingham and Lloyd-Jones (1957) reported that FeEDTA was
rapidly adsorbed on calcareous clay soil. Most of the EDTA remained
water extractable but the Fe precipitated after being exchanged for Ca.
Hemwall (1958) agreed that clay minerals do not retain EDTA but will
precipitate Fe from FeEDTA. Moawad (1970) studied extractability of
FeNaEDTA from four different soils and found that the more acid the
IjOl
-------
soil the longer the Fe remained water soluble.
Cheronis and Schatz (1958) commented that EDTA was known to be very
resistant to degradation by soil microfauna and -flora. Bunch and
Ettinger (1967) tested the ability of sewage to degrade EDTA. Over a
three week period there was little or no apparent degradation of concen-
trations ranging from 5-20 mg/1. Rudling (1972) analyzed for EDTA in
samples from several sewage works and found that the content was the
same in the effluent as in the input - indicative of no degradation.
B. Environmental transport
Knuttson and Forsberg (1966) applied CrEDTA to columns of 20 miner-
als, 5 rock types, and 6 soil types. There was no absorption on quartz,
feldspar, calcite, dolomite, and some micas. Clays, chlorite, Fe(II)-Mg
silicates and other Fe minerals retarded the EDTA somewhat. Nishita
and Essington (1967) studied the movement of EDTA through five soils
of widely different chemical and physical properties. Moawad (1970,
pp 91-100) did a similar study on FeNaEDTA.
C. Bioaccumulation
No indications of bioaccumulation or concern about it were found.
X. TOXICITY
A. Human
Clarke et al (1955) reported on the side effects of human EDTA
therapy. Almost two dozen patients received 10-100 doses of Na£- or
K2EDTA, 5 gin, delivered i.v. in 500 cc of 5% glucose or normal saline
over a 1 1/2-3 hour period. The use of the K salt was terminated be-
cause of intolerable burning sensations at the puncture site and down-
stream. The Na salt also caused burning but at a tolerable level.
Other effects included nausea, diarrhea, dermatitis.
(4 02
-------
Vinerga (1956) reported that therapy using "non-consecutive" daily
doses of 1.5 g CaNa2EDTA had the potential of causing sensitization to
the EDTA, and, consequently, was not recommended.
Kabakow and Brothers (1958) commented that they were able to find
only one literature report of EDTA-caused human fatality. In this in-
stance two patients received daily doses of 28-40 g, apparently dying
from kidney failure.
Meltzer et al (1961) reported the results of 2,000 Na2EDTA treat-
ments involving 81 patients. In Table X is their patient-treatment dis-
tribution. In Table XI is the actual number of occurrences of the in-
dicated side effects (incidences of these side effects had been reported
in Seven and Johnson (I960)). A 3 g dose in 500 ml of normal saline or
glucose solution was administered over 2 1/2-3 hours. Doses were given
every other day until 20 had been given; then a 6-8 week period of no
treatment was initiated before further treatment, if any.
Foreman (1963) reviewed literature reports on side effects from
EDTA therapy.
TABLE X. -DISTIUnUTION OF PA-
TIENTS ACCORDING TO TOTAL NUM-
BER OF INFL'SIONS
Number of Total Number
Patients of Infusions
19 1-10
30 11-20
19 21--10
9 41-60
1 61-80
3 81-120
Tables X - XI reprinted with permission from Am. J.
Med. Sci.. 242:11-17 (1961). Copyright by Charles
B. Slack, Inc.
-------
TABLE IT-SIDE EFFECTS OF EOT A ADMINISTERED
WITH 2000 INFUSIONS
Side Effect
A.
B.
C.
D.
E.
F.
G.
H.
I.
J-
K.
RENAL DAMAGE (increase in BUN, decrease in
PSP excretion, cylindruria, hcmaturia, or
pefiiitont alhummuria greater than 1 +
BURNING AT INJECTION SITE or along course
of \vn:
InitulK only,
Tliroi:0'Kout infusion,
THROMbOIMU.F.KlTIS
HYPOTENSION: Mild: a drop of systolic pres-
sure of 20 mm. without symptoms
Moderate: a drop of systolic pressure of
30 mm. \\ith or without symptoms
Severe: a drop of 30 mm. or more with
distinct hypotcnsuc symptoms
HYPOCALCFAUA: Mild: numbness, tinkling at
circunior.il area or leg cramps or muscle
spasm
Severe: signs of tc-lany (ChvosteVs
sign, and others) or fall in scrum cal-
cium to 7 mg./lOO ml.
SYSTEMIC REACTIONS (febrile reaction, ma-
laise, fatigue, headache, anorexia)
HISTAMINE-LIKE REACTION (sneezing, lac-
rimation, nasal congestion)
ANEMIA or other hematopoictic changes related
to treatment
GLYCOSURIA OR IIYPEKGLYCEMIA
DERMATITIS (pres\imal>ly due to pyridoxine
deficiency)
NAUSEA -r VOMITING: Mild
Moderate
Severe
ABDOMINAL CRAMPS OR PAIN
Frequency of
Occurrence
0
V
30
63
1
8
23
2
20
0
0 . •
0
0
0
0
15
1
2
2
Raymond and Gross (1969) found that EDTA at the level used as a
preservative in ophthalmic solutions was responsible for some cases of
acute allergic conjunctivitis and periorbital dermatitis. They also
found evidence for cases of delayed hypersensitivity.
-------
B. Birds and Mammals
Coune and Driggers (1954) gave male chickens doses of Na^EDTA rang-
ing from 50-200 rag/Kg i.v., i.p., i.m., or s.c. Only the i.v. method,
at 200 mg/Kg, proved fatal. Slow, two minutes, or rapid, 30 seconds,
injection made no difference. Death was attributed to lowering of serum
Ca.
Toyota and Shibata (1956) reported LD-50 values for EDTA salt in
mice as 20.5 mg/Kg oral and 2.6 mg/Kg i.p.
Shibata (1956) reported LD-50 values for EDTA salt in the rabbit as
47 mg/Kg i.v. and 2.3 g/Kg oral; however, extending the i.v. delivery to
10 minutes resulted in no fatalities. In tests of chronic toxicity it
was found that 1 g/Kg orally for one week was fatal, but 1/2 g/Kg for
one month was non-fatal. Also non-fatal was a daily i.v. dose of 20
mg/Kg as a 5% solution.
Shibata (1957) reported the i.p. LD-50 values in mice for CaEDTA
as 7,600 mg/Kg, and for PbEDTA as 7,500 mg/Kg.
Kocher et al (1958) found that the LD-50 in mice for CdNa2EDTA was
63 mg/Kg i.p.
Kocher et al (1959) found that the LD-50 in mice for NiNa2EDTA was
1,244 mg/Kg.
Paulet et- al (1959) found that the LD-50 in mice for Co2EDTA was
50 mg/Kg i.v.
Eybl et al (1959) found that the LD-50 in mice for CoNa2EDTA was
1,948 mg/Kg i.p.
Sykora et al (1960) found that the LD-50 in rats for MnNa2EDTA was
1,930 mg/Kg as a 10% solution i.p. with a ten-day observation period.
-------
Toyoda (1960) found the following l.v. LD-50's in mice: Nai+EDTA 60
mg/Kg, Na2EDTA 460 mg/Kg, CaNa2EDTA 3,250 mg/Kg.
Nofre et al (1962) found the LD-50 in mice for FeNa2EDTA was 281
mg/Kg i.p, in 30 days.
Kostial et al (1962) found the LD-50 in adult female rats for EDTA
was 397 mg/Kg i.p. in one day, or 350-450 mg/Kg with a 95% confidence
limit. Symptoms were severe within 10 minutes and consisted of distress
signs and hypocalcemic convulsions, with death normally occurring in two
hours.
Nofre et al (1963) reported LD-50's for rapid i.p. dosage in adult
male mice of Na2EDTA-2H20 and various MNaEDTA's. The 30-day values, in
order of increasing toxicity, were (mg/Kg): CaNa2 5,351; MnNa2 2,335;
CrNa 2,034; PbNa2 1,678; CoNa2 1,376; NiNa2 589; ZnNa2 519; Na2'2H20
298; FeNa2 281; AWa 183; FeNa 139; CdNa2 31; CuNa2 13; HgNa2 7. Com-
paring the LD-50*s (from a therapeutic viewpoint) of the metal chelates
with the metals alone it was found that, on a weight of metal basis,
the Al, Cu, and Hg chelates were more toxic, the Fe(II) and Fe(III)
chelates were equally toxic, and the others less toxic.
Oser et al (1963) reported oral LD-50's for CaNa2EDTA in fasted
animals as 7 g/Kg in rabbits, 10 g/Kg in rats, and 12 g/Kg in dogs.
Osanai et al (1964) allowed mice to drink water containing varying
amounts of CaNa2EDTA. At 4% the mice died in one week suffering from
diarrhea; at 2% the mice died in seven weeks suffering from anemia; at
1% the mice survived at least 21 weeks suffering only slight, anemia.
There did not appear to be any nephrotoxicity.
Bekemeier (1965) found that the s.c. LD-50 for Na2EDTA in mice was
far from being even relatively constant over a one year period. The
U06
-------
results are reproduced in Graph III.
Graph III. Subcutaneous LD-50 of Na2EDTA as a Function
of the Time of Year
Mg/Kg
3UU
mo
100
ISC
300
ISO
>m
\ '
- I I
j]1
1
MTI
^ -1
-
I
.i l.^_L I—-J... L. -K.,1 ..!,,!
T
1
T
i
i
Cier and Abecassis (1966) studied the genesis in adult male mice of
diabetes mellitus by Na2EDTA and ZnEDTA. A 40 mg/Kg i.p. dose of Na2EDTA
caused symptoms to appear after five days; these were gone after 31 days.
The highest dose tested, 300 mg/Kg, did not elicit symptoms until 17 days,
but they persisted after 31 days in 45% of the animals. Giving 30 mg/Kg
doses on three consecutive days proved to have additive effects. A
40 mg/Kg dose of ZnEDTA gave the same results as that amount of Na2EDTA
in a one week period, but then the glycemia rapidly regressed.
Fiedler (1969) reported that CuNa2EDTA had a higher toxicity than
Cu(II), or CaNa2EDTA, in guinea pigs, rabbits, and rats. Within eight
hours of an i.v. injection of 12.7 mg/Kg in rabbits of CuNa2EDTA there
were 30-250 fold increases in the serum plasma activities of these en-
zymes: alanine and aspartate aminotransferase, glutamate, sorbitol and
lactate dehydrogenase, and fructose diphosphate aldolase. Serum levels
of K increased, Ca decreased, and Na didn't change. In all three species
blood sugar fell to a very low level (after an initial rise in guinea
pigs) .
hoy
-------
Wynn et al (1970) found that rats fed diets consisting of 5 and 10%
Na2EDTA suffered 20 and 60% mortalities, respectively, with the first
death in the higher group occurring in the third week (the study ran for
13 weeks).
Lenza (1971) reported an i.v. dose of 25 mg/Kg of SbEDTA. was lethal
to dogs, causing extreme diastole of the heart.
Ishmel et al (1971) gave sheep single s.c. doses of CaCuEDTA and
found that 18 mg/Kg was the LD-50 in three days. Post mortems showed
excessive fluid in serous cavities, edema of the lungs, mottled livers,
congested kidneys, and subendocardial hemorrhage. Histological examina-
tion showed hepatic centrilobular congestion, hemorrhage and necrosis.
Swenerton and Hurley (1971) fed pregnant rats diets containing 2
or 3% N32EDTA through all or part of the gestation period. At the lower
level for the whole period litter size was normal, but the newborn were
smaller than normal and 7% were malformed. At the higher level for the
whole period all fetuses had been resorbed. At the higher level for the
6-21 day portion litter size was less than half the normal, all newborn
were very small and all were grossly malformed. Results similar to the
6-21 day period were seen when the higher dose was given during the 6-14
day period.
C. Lower Animals
van Asperen and van Esch (1956) injected cockroaches with 30 yl of
67 mM EDTA solution. Within one day there was 30% mortality (the remain-
ing insects recovering). In the initial 15 minutes appeared symptoms of
paralysis and intoxication, all traces of free Ca in the haemolymph having
disappeared.
Khristolyubova (1961) incubated Drosophila eggs in a nutrient medium
108
-------
containing EDTA. Mortality in two-three days was 50%. All hatched
adults were stunted. An above normal number of nucleoli was present in
the salivary gland chromosomes.
Terriere and Rajadhyakshia (1964) found that spider mites produced
fewer offspring when feeding on leaves treated with various EDTA metal
complexes.
Ulitzur and Shilo (1966) reported an LD-100 for minnows immersed in
a 0.3 mM EDTA solution (about 80 ppm).
Sell and Schmidt (1968) reported that concentrations in the diet of
cabbage loopers of as low as 0.05% EDTA delayed development and caused
developmental aberrations on occasion; at 0.5% pupation was completely
suppressed.
Brahmarchary et al (1968) found that 5mM EDTA stopped cleavage of
eggs of Lymnaea (fresh-water snail) after one hour exposure.
Noble (1970) reported that cell aggregation and change from bladder
to filiform amebocyte in the sea cucumber was prevented by the presence
in the water of EDTA at pH 6, but at pH 7.8 only the cell aggreation
didn't occur.
D. Plants
Sussman (1954) found that ascospores of Neurospora tetrasperma
were inhibited at the germinating stage when in the presence of 3.5 mM
EDTA, but when dormant or only newly activated were insensitive to much
higher concentrations. It was demonstrated that the EDTA was not
penetrating into the spore.
-------
Eversole and Tatum (1956) found that different strains of an alga
either incurred increased mutation from contact with EDTA prior to
mating, or were unaffected.
Shannon and Mohl (1956) found that EDTA at 800 ppm in a nutrient
solution was toxic to bush beans after three weeks.
Delaunay (1958) found that EDTA caused chromosome crossovers in
spores, and seemed to stabilize chromosomes broken by x-rays.
Marlatt (1959) studied the effect on lettuce of various metal-EDTA
complexes applied in different ways. Yields were reduced by soil appli-
cation of 224 Kg/hectare of Ca- or ZnEDTA. Spraying the plants with
2.3 g/1 of FeEDTA burned them, and with 4.6 g/1 of ZnEDTA killed some.
Michaelis and Rieger (1963) reported that immersion of the roots
of Vicia faba in 1 mM EDTA for 20 hours produced 3.5 times as many
chromatid aberrations as normal.
Baranauskaite and Rancelis (1966) soaked horse beans for 15 hours
in 0.02 or 0.2% EDTA solutions before soil planting. At the higher con-
centration germinating ability and rate of germination were reduced.
Tsarapkin (1966) confirmed Delaunay's finding (above) that EDTA
stabilized fragmented chromosomes, which can lead to increased mutation.
Rancelis and Luksa (1967) found that chlorophyll mutations were
present in the 2nd and 3rd generations of horse beans which had been
treated with 0.02-0.2% EDTA.
In a study of the effect of EDTA on nuclear division in Triticum
vulgare, Retezeanu (1968) found that a concentration of 9 mM was suffi-
cient to fragment the chromosomes during anaphase and telophase.
Matsuda (1968) found that EDTA at over 5 ppm retarded root growth
IjlO
-------
and decreased yield of rice plants.
Dumitrescu and Retezeanu (1970) found that roots of Lupinus albus
treated with 1 mM Na2EDTA suffered from nuclear fragmentation.
Joshi and Patil (1971) treated Bryophyllum pinnatum with 0.05-20.0
mM Na2EDTA, and then allowed C-14 labelled C02 to be incorporated in
sunlight for one hour. Analysis of the plant for various compounds and
amount of C-14 therein gave the results in Table XII. Plants in 20 mM
solution died in a few days.
TABLE ffl., EFFECT OF F.DTA ON THE DISTRIBUTION OP
RADIO.VCTIVITY IN DIFFF.UT.NT FRACTIONS FOLLOWING
"CO, LIGHT FIXATION IN LEAVES OF D. pinnalnm
(Values of incorporation of radioactivity in individua1 com-
pounds are expressed as percentage of total activity counted on
chromatograms while the rate oC fixation, is expressed as
counts/min/nig fresh tissue)
Compound
Glucose
Fructose
Sucrose
Total
Control
50 tiAT
SUGARS
9-9 2-31
4-57 1-64
34-19 43-54
48-66 47-49
EDTA
0-005M
2-69
1-73
1-92
6-34
COnc.
0-OUf
1-18
—
0-36
1-54
0-02M
1-92
9-2
1-02
12-12
SUGAR PHOSPHATES
Sugar diphos-
phntc
Sugar mono-
phosphate
PliospUoenol
pyruvate -f-
2-76 0-48
— - 0-6
— 2-67
0-32
0-64
1-6
Oil
—
005
0-S1
076
1-27
piiosphojjlyceric
acid
Total
2-76 3-75
2-56
0-16
254
AMINO AC-IDS
Aspartitc
Gliitamate
fViciiie-scrinr
AJannic
Thri-omne
Lcucinos
Total
13-52 2-55
7-71 0-91
1;51
50! 6- 13
—
— 2-8S
2627 14-28
1-47
2-56
2-94
5-12
4-98
1-53
IRdO
0-11
015
0-55
0-67
—
045
1-93
2-81
2-3
6-39
31-84
—
17-38
60-72
ORGANIC ACIDS
Citrate
Maiatc
Siicrinate
Fnm.irate
Total
Kate of fixation
15-62 16-7.1
1-52 t-r,7
1-61 15-5S
3 23 0 66
21-93 34-64
200 221)
62-33
07
1-08
2-24
67-35
189
93-13
1 -
-------
E. Micro-organisms
Ujiie (1959) found that at pH 6-8 EDTA was not toxic to E. coli
over 48 hours of contact, but it did inhibit propagation.
Nezval (1964) found that EDTA was synergistic with the bacteriocide
Septonex against Pseudomonas aeruginosa. On a concentration basis a
solution containing one part of Septonex to two of EDTA was ten-fold as
effective after five minutes exposure as a solution containing only the
same amount of Septonex.
Patel and Shah (1965) tested the antifungal and bacterial activity of
2% Na2EDTA against that of penicillin, streptomycin, and various chemicals
of simpler structure. Against nine gram-positive bacteria, the EDTA was
about 60% as effective as 5 yg/ml of the K salt of penicillin; against
three others the EDTA was at least as effective. Against six gram-
negative bacteria, the EDTA was about 80% as effective as 20 yg/ml of
streptomycin sulfate; against four others the EDTA was 100-130% as
effective. Against 0.25% methyl paraben the EDTA was more effective in
eleven of twelve fungi tested; against 0.5% benzoic or salicylic acid
the EDTA was slightly more effective in eight of twelve (not the same
eight).
Goldschmidt et a.1 (1967) found that the male strains of E. coli were
far more sensitive than the female to a mixture of EDTA and Tris.
Neu (1969) could not completely confirm Goldschmidt's results,
finding closer toxicity between male, female, and Hfr strains.
Nezval and Ritzerfeld (1970) found that EDTA was synergistic with
chloramphenicol or neomycin, but not with carbenicillin or gentamycin,
against Pseudomonas aeruginosa.
Russell (1971) reviewed the antibacterial activity of EDTA.
-------
XI. CURRENT REGULATIONS
No information other than the FDA limitations given in Section
III was found.
XII. STANDARDS
No information was found.
-------
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llh
-------
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15
-------
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U16
-------
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• 17
-------
Knutsson, G., and H. G. Forsberg (1966). Laboratory Evaluation of
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l(20
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U.S. Tariff Commission, Synthetic Organic Chemicals, U.S. Government
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U2U
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FORMALDEHYDE RESINS
SUMMARY AND CONCLUSIONS
The finished products made from the thermosetting resins resulting
from the reactions of formaldehyde with urea, melamine, or phenol are
infusible, insoluble, hard and mar-resistant, flame-resistant, and are
chemically inert under use-related conditions.
The finished products made from the thermoplastic resins resulting
from the reactions of formaldehyde or trioxane with ethylene oxide have
high strength and rigidity, good electrical properties, abrasion resis-
tance, good flame resistance, and are chemically inert under use-related
conditions.
The expected trend in the production of the formaldehyde resins is
upward. Some set-backs in the production and sales of the formaldehyde
resins have been experienced because of the energy crisis and shortages
of starting materials, and because of a decrease in demand from some
manufacturing areas. These set-backs are considered by the plastics
industry to be cyclical, however, and the overall trend in the manu-
facture and sales of these resins is expected to be upward. In fact,
an annual growth rate of 6.1% per year has been predicted by the
plastics industry, from the present time to the year 2000.
The versatile formaldehyde resins have usually wide application
ranges. The amino resins are used in closures and wiring devices, large
and small appliance housings, dinnerware, buttons, ash trays, and
utensil handles. They are used as adhesives in plywood and in lami-
nating. In textile treating, they are used for greaseproofing, water
U25
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repellancy, and flame retardance. In paper treating, they are used for
improving wet strength, rub resistance, and dry tensile values.
The acetal homopolymer is used to replace metal parts in the
plumbing industry. It is used in truck-trailer connectors and in
such a variety of items as furniture casters, hardward items, bodies
of lighters, replaceable cartridges in shavers, toy components,
telephone pushbuttons, and stereo-tape and cassette components.
The acetal copolymer is used in automotive gears and fuel-
emission systems. It is also used to give satiny surfaces and hardness
to pen barrels and other items where an attractive appearance is desired.
Its dimension stability qualifies it for use in aersol containers under
continuous pressure.
The phenolics are used as adhesives in the wood particle board
used in building panels and furniture, and as a water-resistant glue
for exterior grade plywood. They are used extensively as automotive
components in transmissions, distributor caps, coil towers, rotors,
fuse blocks, for brake linings, clutch parts, and transmission bands.
Because of the tremendous variety of uses that have been found
for the formaldehyde resins, it is almost impossible to avoid daily
contact with products which have been manufactured from them. The
fact that there are no reports in the collected literature concerning
toxic effects from contact with any of these products would certainly
verify that the formaldehyde resins are physiologically inert in the
finished state.
They are not biodegradable, and they persist in their solid form
under normal atmospheric conditions.
1426
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During resin manufacturing, however, certain starting reagents,
fillers, and resin dusts present a real hazard to workers who are without
adequate protection. While urea and melamine have no history of toxicity,
nor have they been known to be a source of occupational problems, formald-
hyde, phenol, and asbestos ( a filler) are toxic. Formaldehyde is a
sensitizing agent and a mucous membrane irritant. Phenol is highly cor-
rosive to the skin and produces severe burns. Asbestos, if introduced into
the respiratory tract, causes emphysema and neoplasms of the lung. Granu
lomas were found in the lungs of rats which had been subjected to the
inhalation of the dust of acetal resin; granulomas were also found in tl
subcutaneous tissue and in the peritoneal area of rats after acetal
powders had been injected at these sites. All of the reports in the
literature which dealt with toxic symptoms in workers were written outside
of the United States.
In fact, in all of the articles written in the United States concern-
ing the safety of workers in the plastics industry, the stress was placed
on equipment safety. This situation is about to reverse, however,
because of the recent indictment of two chemicals which are used in
industry, and with which carcinoma has been associated. Formaldehyde is
directly related to one of these chemicals - chloromethyl methyl ether,
which has been shown recently to cause malignant lung neoplasms.
Formaldehyde in contact with hydrochloric acid will yield chloromethyl
methyl ether. Conditions for this reaction were not stated in the cursory
news medial report (when formaldehyde and hydrochloric acid are used for
chloromethylation in the laboratory, a zinc chloride catalyst is used).
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The question might be raised here as to whether workers in the United
States have been well protected by clean factory operations and by com-
pliance with the emission limits which have been set for basic raw materials,
resins, and compounds, or whether the toxic hazards have been known, but
experience and clinical investigation are just now producing sufficient
documentation to put the hazards into proper perspective.
128
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FORMALDEHYDE RESINS
I. PHYSICAL PROPERTIES
A thermosetting resin is a crosslinked, polymeric material
which has been rendered substantially infusible and insoluble by
curing it with heat or with chemical catalysts. Urea-formaldehyde
resins, melamine-formaldehyde resins, and phenol-formaldehyde
resins are included in this group.
A thermoplastic resin is a material with a linear macromolecular
structure which will repeatedly soften when heated and harden when
cooled. The acetal homopolymers and the acetal copolymers are inclu-
ded in this group.
A. AMLNO RESINS
Amino resins are thermosetting condensation polymers formed in
the reaction between formaldehyde and organic compounds which con-
tain more than one -NH2 group per molecule. Urea-formaldehyde resins
and melamine-formaldehyde resins are the most commercially significant
compounds of this group.
The physical form of these reaction products may be either
fluffy powders or dense granules. Specific fillers may be added
to meet specific requirements. The addition of alpha cellulose, for
instance, enhances strength, moldability, and dimensional stability.
Alpha cellulose-filled compounds are translucent. The addition of
this filler to urea and melamine molding compounds yields moldings
with an attractive gloss. They are quite hard and mar-resistant.
Molded items do not collect dust by build-up of a static electrical
-------
charge and so do not require the addition of an antistatic agent.
Both urea and melamine compounds are resistant to oils, solvents, and
greases. They are intrinsically flame-resistant and have good elec-
trical arc resistance. They exhibity high-heat distortion temperatures,
good strength properties, and are odorless and tasteless.
Substitution of alpha cellulose by other fillers enhances
specific physical or electrical properties; this is usually at the
expense of appearance, handling properties, or molding properties, how-
ever. Wood flour, cotton fabric, asbestos, and glass fibers are al-
ternative fillers for these compounds.
B. ACETAL RESINS
Acetal resins are thermoplastic resins containing the following
repeating unit: -CH2-0-.
These resins are produced from formaldehyde or trioxane (a cyclic
trimer of formaldehyde), either as homopolymers of formaldehyde or as
copolymers of trioxane with other organic compounds (e.g., ethylene
oxide).
High strength and rigidity, dimensional stability, and resilience
are some properties of these compounds. Acetal homopolymer.'s are avail-
able in a number of compositions to fit a variety of end-use require-
ments. These compositions differ primarily in melt viscosity. Mechan-
ical properties of the various grades are similar except for tensile
elongation and Impact strength. Acetal homopolymer has a tensile
strength at room temperature of 10,000 p.s.i. with no true yield point
and a flexural modulus of 410,000 p.s.i. Acetal homopolymer has out-
' <
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standing creep resistance (creep is a time-related change in dimension
of a material under load). Its fatigue endurance at room temperature
is 5000 p.s.i. It is resistant to organic solvents, although contact
with strong acids or strong bases is not recommended. It has good
abrasion and frictional resistance with hardness and resistance to
scratching. It maintains good electrical properties under high tem-
perature and humidity exposure, after immersion in water, and on aging.
Acetal copolymer compares with die-cast metal in its resistance
to creep under load at elevated temperatures. It has excellent elec-
trical properties, low moisture sensitivity and high solvent and alka-
li resistance. It is attacked by oxidizing agents and acids, however.
Samples in boiling water retain nearly original tensile strength for
six months, but for maximum long-term continuous use, the recommended
temperature in water is l80° F.
C. PHENOLIC RESINS
Phenol and aqueous formaldehyde are reacted in the presence of
alkaline or acid catalysts to produce both liquid and rigid resins
(the phenol-formaldehyde resins). These have excellent dimensional
stability, heat resistance which is superior to most other thermo-
setting materials, high heat-deflection temperature, outstanding creep
resistance, and good flame resistance. Almost all phenolics for
years have been rated nonburning according to ASTM D635. More recently,
certain phenolics received formal self-extinguishing Group I ratings
according to Underwriters Laboratory Bulletin 94. A further
discussion of flammability ratings is given in Section XII, B,
General purpose materials with wood flour as the main filler are
used im most applications where the basic property profile of phenolics
-------
is adequate. Where higher heat-resistance is required, the wood
flour is replaced by mineral-filled compounds (such as asbestos) .
D. VINAL FIBERS
Fibers based on the reaction product of polyvinyl alcohol and
formaldehyde are known as vinal, vinylon, or PVA fibers. Fabrics
made from these fibers have a cotton-like feel. They are strong,
abrasion resistant and moisture-absorbent, and are quick-drying and
inexpensive. However, they cannot be dyed in bright colors, they
are susceptible to shrinkage, and they cannot be heat-set. Vinal
fabrics soil easily, have poor elasticity, and wrinkle readily. It
is for these reasons that vinal has not been successful as an apparel
fiber. However, it has industrial applications because of its high
strength, durability, and resistance to weathering, heat, and abrasion.
The fibers have excellent adhesion to plastics, usually without the
need for coupling agents. They are light-weight with a density of
1.25. They do not break under high pressure and shear of injection
molding (Modern Plastics Encyclopedia, 1973-74; Chemical Economics
Handbook).
II. PRODUCTION
A. AKLNO RESINS
Some of the leading manufacturers of amino-formaldehyde resins in
the United States are American Cyanamid Co. (Cymel, Urac, Melurac);
Allied Chemical Corp. (Plaskon); Rohm and Haas Co. (Uformite, Phonite),
and Borden Chemical Co. (Casco).
h32
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The following production figures have been calculated from data
reported by several sources.
ESTIMATED PRODUCTION FIGURES
(Metric Tons)
UREA RESINS MELAMINE RESINS
1964 190,000 69,000
1965 225,000 78,000
1966 250,000 95,000
1967 234,000 97,000
1968 315,000 94,000
1969 343,000 95,500
1970 321,000 117,700
In 1971, urea and melamine sales increased by about 15%
over 1970. Their combined sales again increased by 15% in 1972.
The sales figures for 1973 show an increase of 13% over 1972, which
is a 2% drop from the previous year's growth rate. Further production
figures for urea-formaldehyde resins and melamine-formaldehyde resins,
based on consumption and applications, are included in Section III,
USES.
B. ACETAL RESINS
Acetal resins are produced in the United States by two companies:
Celanese Corporation, Celanese Plastics Company Division, Bishop,
Texas; and E. I. duPont de Nemours and Company, Inc., Plastics Depart-
ment, Parkersburg, West Virginia, according to Chemical Economics
Han db ook.
The trade name of the Celanese product is Celcon, which is an acetal
copolymer. Plant capacity was estimated at about 70 million pounds
per year in mid-1971.
-------
The trade name of the duPont product is Delrin, which is an
acetal homopolymer produced in several grades. Plant capacity was
estimated at about 55 million pounds per year in mid-1971.
Since there are only two producers, separate data on the U.S. pro-
duction of acetal resins have not been published by the U.S.. Tariff Com-
mission. The following estimated production figures are based on
consumption estimates made by trade sources.
ACETAL RESIN PRODUCTION (ESTIMATED)
(Metric Tons)
1965
1966
1967
1968
1969
1970
20 ,000
26,000
28,000
33,500
38,900
39,000
In 1971, acetal resin sales increased 10% over 1970 sales. The
1972 sales increased 11.5% over 1971, and the 1973 sales attained a
record advance of 18.5% over 1972.
Additional statistics for the acetal resins, based on consumption
and applications, are given in Section III, USES.
C. PHENOLIC RESINS
Among the manufacturers of phenol-formaldehyde resins in the
United States are Ashland Chemical Co. (Arofene, Arochem, Arotap),
Borden Chemical Co., Clark Oil and Refining Corp., Firestone Tire and
Rubber Co., Formica Corp., General Electric Co., Hooker Chemical Co.,
Rohm and Haas Co., Westinghouse Electric Corp., Union Carbide Corp.
(Bakelite), and Monsanto Co. (PF-535; Resinox 517).
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PHENOL-FORMALDEHYDE RESINS PRODUCTION (ESTIMATED)
(Metric Tons)
1962
1963
1964
1965
1966
1967
1968
1969
1970
1974
263,400
284,000
322,200
356,600
405,900
384,000
432,200
464,800
462,200
212
212 (preliminary
figures, May)
In 1971, the sales of phenol-formaldehyde resins remained on a
level with 1970. In 1972, new molding techniques led to an increase
in the sales of phenolics of 30% over 1971; the greatest growth was
in the field of appliances. Phenolics showed only a 0.4% gain in
1973 over 1972, attributable to a major drop in the plywood market.
Additional data on the production of phenolics is given in Section
III, USES.
D. VINAL FIBERS
Vinal fibers are not produced in the United States. Approximately
103 metric tons were consumed in 1970, all of which were imported.
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III. USES
A. AMINO RESINS
The properties of the alpha cellulose-filled urea molding materi-
als qualify them for use in closures, wiring devices, electric blanket
controls, toilet seats, stove and refrigeration hardware, knobs,
buttons, and electric appliance housings. The closure applications
(both straight-wall and reverse-taper types) and wiring devices
(switchplates, toggles, receptacles) predominate. Allied Chemical
Company's Plaskon is a typical urea resin recommended for these
applications .
Alpha cellulose-filled molding compounds are used to form dinner-
ware, buttons, ash trays, utensil handles, electric shavers and housings.
American Cyanamid Company's Cymel 1077 (a melamlne-formaldehyde
resin) is a representative of this group of resins.
Adhesives are manufactured from both urea and melamine resins, but
the bulk of these are urea-formaldehyde resins. The melamine adhe-
sives are superior to urea adhesives in water resistance and weathering,
giving boil-resistant bonds.
As laminates , the melamine resins offer superior hardness and
wear resistance; in industrial laminates their added advantages are
flame, arc, and heat resistance. Some of the applications are in the
manufacture of tabletops, countertops, and wall paneling.
The amino resins are used in textile-treating ia creaseproofing,
shrinkage control, stiffening, water repellency, and flame retardance.
In paper treating, the amino resins improve wet strength,
burst strength, rub resistance, and dry tensile values.
-------
Alkylated methylolureas and methylolmelamines are extensively
used with alkyl resins in baking enamels for greater hardness, mar and
chemical resistance, and durability (Modern PJLasjzics Encyclopedia,
1973-74).
Following are consumption statistics for urea and melamine resins
in metric tons:
1971 1972 1973
Urea Resins
Closures 6,900 6,800 7,900
Electrical devices 11,400 10,900 12,600
Melamine Resins
Buttons 900 800 900
Dinnerware 19,100 18,200 19,100
Sanitaryware — 500 600
Collective statistics
Bonding and adhesive resins
for:
Fibrous and granulated wood 199,000 232,000 262,000
Laminating 22,000 24,000 24,000
Plywood 31,000 40,000 40,000
Paper treating and coating 14,000 16,000 22,000
Protective coatings 19,000 28,000 33,000
Textile coating and treating 23,000 23,000 26,000
B. ACETAL RESINS
The properties of the acetal homopolymer makes it suitable for
use in the plumbing industry in shower heads, valves, and fittings,
replacing brass and zinc parts. It is used in truck-trailer connectors
and furniture casters. Handles and other hardware items are formed
from the homopolymer, as are the bodies of lighters, replaceable cartrid-
ges in shavers, toy components, telephone pushbuttons, lawn sprinklers,
1437
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stero-tape cartridge and cassette components.
The acetal copolymer Is used in automotive components such as
gears and fuel-emission systems. Since it is noncorrosive in long-term
hot-water exposure and can be used with metals, it finds application
in the plumbing industry. Its creep resistance qualifies it for use
in aerosol containers under continuous pressure. It is used to form
pen barrels and other components where satiny surfaces, hardness, and
stain resistance give added value and better appearance (Modern Plastics
Encyclopedia, 1973-74).
Following are consumption statistics for the acetal resins in
metric tons:
1971 1972 1973
Appliances 4,820 5,600 5,900
Consumer products 3,090 3,500 3,800
Electrical/electronics 1,910 2,100 2,500
Machinery parts 2,550 2,800 3,900
Plumbing and hardware 2,550 2,800 4,000
Sheet, rod, tube 1,320 1,500 1,800
Transportation 6,050 5,700 6,500
The phenol-formaldehyde resins are used in power-brake and auto-
matic transmission components, distributor caps, coil towers, rotors,
fuse blocks, and connectors. They are used to bond friction materials
for automotive brake linings, clutch parts, and transmission bands.
They serve as binders for wood particle board in building panels and
furniture, as water-resistant glue for exterior grade plywood, and as
the bonding agent in acoustical and thermal insulation pads.
-------
Following are consumption statistics for the phenol-formaldehyde
resins in metric tons:
1971 1972 1973
Bonding and adhesive resins for:
Coated and bonded abrasives 8,000 9,100 11,200
Fibrous and granulated wood 30,000 40,000 42,000
Friction materials 14,000 13,400 14,700
Foundry and shell moldings 39,000 43,600 50,000
Insulating materials 88,000 107,000 112,000
Laminating
Building 31,500 26,100 26,200
Electrical/electronics 7,000 7,300 7,300
Furniture 12,000 16,000 17,000
Plywood 152,000 163,000 125,000
Protective coatings 10,000 9,600 10,100
Molding compounds
Appliances 17,500 31,800 41,400
Business machines 4,400 6,100 6,800
Closures 9,100 4,500 4,100
Electrical controls and
switches 40,400 56,000 61,000
Telephones 9,800 9,300 9,500
Wiring devices 15,600 15,900 16,300
Housewares
Utensils and handles 11,200 14,300 14,700
Machine parts 4,000 4,800 5,100
Transportation — 27,700 30,270
A very successful use for the vinal (PVA) fibers is in injection
molded rail-tie retainers in Japan. PVA polyester laminate is also
being used in greenhouse glazing. P¥A cloth and mat are used as sur-
facing veils to improve impact strength, weatherability, and abrasion
resistance.
139
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The major uses for the vinal fibers in the United States are found
in the manufacture of chemical lace and paper. Consideration is being
given to using vinal fibers as tire cord material.
The estimated consumption of the vinal fibers in the United States
is 226,000 Ibs. annually, all of which is imported.
A. TRENDS
In 1973 urea and melamine rose 13%, a drop of 2% from the previous
year's growth rate, principally due to two factors: demand for plywood,
a market for urea bonding, fell considerably, which resulted in a
urea-in-plywood total over 1000 tons lower than in the year before. The
short supply of phenolics (purchasers turned to urea) kept the figures
from becoming even lower. Expected large growth in melamine molding
powders for dinnerware did not materialize; consumption was at the 1972
level.
Bonding and adhesive resins for fibrous and granulated wood have
continued to be big market performers.
The acetals experienced a growth rate of 18.5% over 1972. Plumbing
and machinery parts predominated. New uses included one-piece tape
spools and aerosol containers.
CBS Records designed the one-piece tape spool for its Mark 2
eight-track tape cartridges to replace a two-piece polystyrene/acetal
assembly with a one-piece part molded of Delrin (duPont) homopolymer.
The phenolics showed a gain of 0.4% over 1972. Their 11.3% increase
in molding powders and advances in bonding and adhesive resins were
offset by a major drop in the plywood sector. Molding powders were in
IhQ
-------
brisk demand for appliances, electrical/electronics, housewares and
machine parts. Closures yielded to thermoplastics. Suppliers see a
distinct trend to greater use for injection over transfer and compres-
sion. In other areas demand for phenolic resins for abrasive, friction,
and foundry applications was up about 15%, reflecting activity by such
major steel users as the automotive industry (Modern Plastics, 1974; 1).
Century growth figures have been projected (in metric tons) as:
from 362,000 metric tons produced in 1971, the production rate of urea
and melamine resins will reach 2,000,000 metric tons in the year 2000,
with an annual growth rate of 6.1%.
The phenolic resins will increase in production from 541,000
metric tons in 1971 to 3,000,000 metric tons in the year 2000, with an
annual growth rate of 6.1% (Modern Plastics, 1973; 7).
IV. CURRENT PRACTICE
There are no particular problems in the storage, transport, or
handling of the formaldehyde resins; the completely polymerized finished
resins are physiologically inert, non-toxic materials.
The problems lie in the manufacturing and in the disposal of
these resins. Most of the starting reagents are toxic and must be
carefully handled. The finished products contain nitrogen, and the
combustion by-products produced by high-temperature incineration often
are more noxious than the plasticizers. The combustion products are
given in Section V, ENVIRONMENTAL CONTAMINATION.
UUl
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V. ENVIRONMENTAL CONTAMINATION
Environmental contamination by the formaldehyde resins occurs both
during manufacturing processes and during disposal processes .
While the completely polymerized finished plastics are derma-
tologically inert, most of the starting products are highly irrita-
ting to the skin and mucous membranes. Where careful precautions are
not used, resin dusts easily contaminate the air in workshops and
might possibly be carried from there into the atmosphere. In. certain
media, small amounts of formaldehyde can be released and can then
oxidize and result in formic acid, a highly caustic and toxic compound.
Formaldehyde, cresylics, and phenols are present in the waste
water from phenolic resin production. In the impregnation of paper
with phenolic resin, low molecular weight resin is driven off during
the impregnation and during the curing process in treater ovens. With
less than perfect precautionary measures, both water pollution and air
pollution will occur from these sources.
The thermo-oxidative destruction of the formaldehyde resins results
in volatile toxic products. In the combustion of phenol-formaldehyde
resins, water and formaldehyde are released at temperatures below
400°C; carbon monoxide, carbon dioxide, benzaldehyde, benzene, toluene,
methane, phenol, and phenolic derivatives are released at temperatures
between 400°C and 600°C; and carbon monoxide and hydrogen are released
at temperatures above 600°C (Dotreppe-Grisard, 1968).
During the combustion of melamine-formaldehyde resins and urea-
formaldehyde resins, the following products result: hydrogen, methane,
acetylene, ethene, ethane, propylene, propane, butene, butane,
-------
methyl alcohol, ethyl alcohol, acetone, acetic acid, furan, formal-
dehyde, methylfuran, dimethyIfuran, benzene, toluene, carbon monoxide,
carbon dioxide, ammonia, and cyanic acid (Hiramatsu, 1967) .
An air purifying device for smokestack mounting has been developed
by Marks Polarized Corp., Whitestone, N.Y. (U.S. Pats. 3,503,704
and 3,520,662). Polluted air is passed through an aerosol composed
of charged water droplets. The device removes 99% of suspended
particles, noxious gases, and other plastics combustion products
(Anon., 1970; 1)
Three Japanese manufacturers have offered solutions to air pollu-
tion from plastics manufacture. Takuma Boiler Manufacturing Co. has
designed an incinerator capable of burning several different types of
plastics simultaneously, without emitting any polluting gases. This
incinerator can handle 100 metric tons/day.
Okumura Kikai's model is smokeless within three minutes after
firing. Forced compressed air allows the furnace to be fired without
a starter. While a variety of plastics can be burned, only one type
of resin can be destroyed in a single load.
Takuma's model burns pulverized plastics at 300°C. The gases given
off are automatically passed into a second furnace and burned at 1000°C.
Then the load is passed through a heat exchanger and then through a dust
collector. The remaining waste is collected in a smokestack
(Anon., 1970; 2).
A large processing line installed at the Narmco Materials Div. of
Whittaker Corp., Anaheim, Calif, converts its own pollutants into a
source of energy for heating and air conditioning. This company pro-
Ui3
-------
cesses glass fabrics impregnated with epoxy or phenolic adhesives .
The thermosetting resin systems allow solvents to evaporate during
heating. The fumes are drawn out of the oven and piped to the incin-
erator section of the heating system, where they are mixed with natural
gas and burned. The intense heat of combustion produces steam in
the boiler section of the system, and the steam is piped back to heat
the air from the curing oven. These fumes provided 20% of the fuel
required for process heat and plant air conditioning.
Emissions consist of carbon dioxide and fall within safety limits
established by the Air Pollution Control District (Hauck, 1971, 1) .
At the Spaulding Fibre Co., Tonawanda, N.Y., a closed-loop
system is used to incinerate all wastes from plastics manufacturing.
The resultant heat is then used to generate steam which powers the
plant. Spaulding produces high-pressure laminates, basic phenolic
resin, and paper (Anon. 1971; 1).
A molding machine that can process 100% of scrap regrind has been
developed by Werner and Pfleiderer Corp., Waldwick, N.J. The machine
(Remaker) will handle film scrap directly, without grinding or other
intermediate conditioning. Virtually any type of thermoplastic
reportedly can be molded by this method (Hauck, 1971; 2) .
VI. MONITORING AND ANALYSIS
A. AKLNO RESINS
The urea-formaldehyde resin content of paper can be measured
in the 0.3% to 3% range by differential infrared spectrometry (Wise
and Smith, 1967). Standards are prepared by adding a known volume
khh
-------
of standard solution to a paper of known weight and calculating the
resin added.
Any contaminants present are removed from the paper test sample
by successive extractions with carbon tetrachloride and methyl
ethyl ketone, using a Soxhlet apparatus. The paper is then placed in a
beaker of acidulated ethanol (4 cc of hydrochloric acid per liter),
heated to just below the boiling point, and decanted after one-half
hour. This process is repeated until the supernatant liquid is
colorless. Hot water is used as a final extractant. The specimen
is air-dried and then oven-dried at 105°C for one-half hour. This
extraction procedure causes no detectable change in the urea-formalde-
hyde resin content of the paper, as measured at 6.05u-
A 1:1 blend of polybromotrifluoroethylene and tetrachloroethy-
lene is used as a coating liquid for both the standard sample and the
test sample. The thickness of both of these specimens should corres-
pond to 0.002 to 0.005 g/cm to permit accurate measurement of the
aliphatic carbon-hydrogen stretching band at 3.4v (the internal
standard). The coating liquid must be equal, in amount and in thick-
ness, on both the sample and the standard specimens.
The specimens are mounted between sodium chloride plates. The
spectrometer is operated at the highest programmed slit width of 1000
to provide a high signal/noise ratio.
Whenever the amount of urea resin is as high as 0.5%, it is
possible to identify the resin by the presence of both major amide
bands at 6.05 and 6.4y.
Melamine resins in wet-strength papers can be detected and
-------
estimated by ultraviolet spectrophotometry (Hirt, King, and Schmitt,
1954) .
The cut-up paper samples are refluxed in 0.1 N hydrochloric acid
to extract and hydrolyze the resin to the melaminium ion which is
then measured spectrophotometrically. Melamine has a strong absorp-
tion near 235 my. This maximum, which is achieved in hydrochloric
acid, becomes a slight shoulder in neutral or alkaline medium, thereby
confirming the presence of melamine. If no band near 235 my is ob-
served in hydrochloric acid, the presence of melamine can no): be
reported.
Braun and Jung (1970) present a simple method for differentiating
between urea-formaldehyde and melamine-formaldehyde resins when both
are present in a test resin sample. The test material is hydrolysed
in concentrated hydrochloric acid by heating the mixture to the boiling
point. An aliquot of the hydrolysate is made alkaline with dilute
sodium hydroxide, and a drop of sodium hypochlorite is added. In
the presence of a urea-formaldehyde condensate, the solution remains
colorless at this point, while carbon dioxide is generated; a
melamine-formaldehyde condensate gives a white precipitate. The
precipitate is filtered and a drop of sodium hypochlorite is added.
A yellow to orange color developing within thirty minutes is indica-
tive of the presence of melamine.
To another aliquot of the hydrolysate, freshly prepared furfurol
reagent is added (5 drops of pure, freshly distilled furfurol, 2 ml
of acetone, 1 ml of concentrated hydrochloric acid, and 2 ml of water) .
The presence of a urea-formaldehyde condensate is indicated by the
-------
development of a yellow to red color; the presence of melamlne has
no effect.
These same authors also describe a thin-layer chromatographic method
for distinguishing between urea-formaldehyde and melamine-formaldehyde
resins. The test sample is hydrolysed in 1 N sulfuric acid for two
hours in a boiling water bath, after which any unhydrolysed portion is
filtered out. The formaldehyde formed is distilled off until no
further reaction is given between the distillate and carbazol/sulfuric
acid. The pH of the test mixture is then adjusted to 6.5 with diluted
barium hydroxide, and the precipitate is centrifuged out. Varying
amounts of the solution, from 3.0 yL are applied to thin-layer plates,
and the results are compared with control samples.
The following Rf values are given when 3 yL of test solution
is applied, using a 15 cm path:
Solvents Melamine Urea
Pyridine:Benzene:Water 0.20 0.27
Acetonitrile:petroleum ether:carbon tetrachloride:
tetrahydrofuran:formic acid:water (80:10:10:10:4:
10) 0.52 0.69
Acetonitrile:chloroform:benzene:methyl alcohol:
water (20:50:30:40:10) 0.31 0.39
Pyridine:benzene:acetonitrole:water (50:50:30:10) 0.44 0.57
Pyridine:benzene:acetonitrile:water (50:50:30:5) 0.12 0.20
Pyridine:benzene:acetonitrile:water (50:80:60:5) 0.23 0.36
Acetonitrile 0.00 0.03
Reprinted with permission from Gummi. Asbest. Kunstat. 23(6), 618,
620, 622, (Ger), (1970). Copyright by A.W. Centner Verlag.
The free formaldehyde present in urea-formaldehyde foams can be
determined by the method of Ardelt and Opel (1962). A foam sample,
10 x 10 x 5 cm, is crumbled under 1600 ml of water and kept for thirty
1*1*7
-------
minutes. A 50 ml aliquot of the supernatant solution is transferred
to a 100 ml volumetric flask containing 10 ml of N sodium hydroxide.
The formaldehyde is then determined polarographically under nitrogen.
The usual adjuncts of the foam do not interfere.
The formaldehyde present in textiles which have been treated with
either urea-formaldehyde resin or malamine-formaldehyde resin can
be determined by first extracting 36 cm2 of material with 250 ml of
water for 24 hours at room temperature. Two ml of sulfuric acid
and a 0.5% soulution of the sodium salt of chromotropic acid is added
to 2 ml of the extract. The mixture is then heated and held at a
temperature of 100 C for 15 minutes. After 30 minutes, the absorbance
is measured at 575 mp. The precision is 2 pg (Vankos, Borza, and
Palfi, 1967).
B. ACETAL RESINS
No analytical techniques for the acetal resins were found in the
literature collected for this study.
C. PHENOLIC RESINS
The phenolic content of resins of unknown origin can be estimated
by a modification of the nitrous acid test for free phenols, which
produces a yellow color that is specific and is colorimetrically
applicable (Swann and Weil, 1956).
A small sample of resin, varnish, or enamel vehicle is weighed,
dissolved in n-butyl acetate, and diluted to definite volume. An
aliquot of the resulting solution, estimated to contain not more than
-------
6 mg of phenolic resin, is transferred to a 250 ml Erlenmeyer flask.
Butyl acetate is added to bring the total volume to 40 ml. Ten ml of
10:1 sulfuric acid (3.6 N) is added, followed by 2 ml of a freshly
prepared 10% aqueous solution of sodium nitrite. The flask is vented
and placed in a 70°C water bath for one hour, during which time
gentle agitation is applied. The sample is then cooled and transferred
to a separately funnel with water. The solvent layer is washed twice
with water. After the final water layer is removed, the solvent layer
is filtered into a 50 ml volumetric flask and diluted to volume.
Colorimetric comparison is made at 425 my, against a blank cell of
water. The phenolic resin content of the sample is determined from
a calibration chart plotted from the results of tests on known
standards.
A rapid gas-chromatographic method for determining phenol-
formaldehyde resin in plywood adhesives was developed by Stevens and
Percival (1964) . A dual gas chromatograph is used. Column A is
copper tubing, twelve feet in length and one-fourth of an inch in
diameter, packed with silicone SF-96 (for phenol) . Column B is
copper tubing, sixteen feet in length and one-fourth of an inch in
diameter, packed with 10% sucrose octoacetate on Teflon 6 (for
formaldehyde). The operating conditions are: detection cell, 250°C;
d.c. current, 200 ma; injection temperature, 250°C; column temperature,
130°C; and helium flow rate at 60 p.s.i.g., 120 ml per minute through
column A and 59 ml per minute through column B.
The phenolic plywood adhesives can be diluted with water and injec-
ted directly into column B for formaldehyde analysis; this, however,
-------
causes plugging of the injector and other mechanical difficulties.
To preclude these difficulties, the resin solids are precipitated
by neutralizing with acid and the aqueous solution is injected.
Ten grams of resin are weighed out, together with the internal
standards: 1-butanol for formaldehyde and m-cresol for phenol. The
sample is then diluted with approximately equal volumes of water and
divided into two fractions. For formaldehyde analysis, one fraction
is acidified with concentrated hydrochloric acid or sulfuric acid with
vigorous stirring. The resin solids are then filtered off, leaving
the aqueous solution for injection into column B. Ten ml of ether is
added to the solid fraction, and the mixture is stirred vigorously while
slowly adding acid. When neutralization is complete, the syringe is
filled from the ether layer for injection into column A.
The peaks are read on a Leeds and Northrup Speedomax Type G
Recorder.
The application of pyrolysis gas chromatography to the analysis
of phenol-formaldehyde resins is reported by Zulaica and Guiochon
(1966). The samples used (0.1-0.5 mg) were in the form most conveni-
ently obtained: powder of resols or irregular fragments oE resite.
The pyrolysis is carried out at temperatures between 700 and
750°C in a conventional platinum coil. The experiments are carried out
on a Perkin Elmer 116 instrument under the following conditions:
the column is nine meters long and four mm in diameter, packed with
4% tri(2,4-xylenyl)phosphate on Chromosorb P (250-315 y) . The working
temperature is 180°C.
The pyrolysis products are detected on a katharometer.
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VII. CHEMICAL BEACTIVITY
The formaldehyde resins, infusible and chemically inert in the
finished state, undergo no transformations under normal, use-related
conditions.
The ami no resins are intrinsically flame-resistant and have
outstanding resistance to chemical solvents. The acetal resins are
unusual among thermoplastics in their resistance to organic solvents,
but they are subject to attack by strong acids and strong bases.
The phenolic resins are considered to be nonburning materials which
are unaffected by most chemicals.
However, the formaldehyde resins are organic materials and all
organic materials will undergo combustion when the proper conditions
are met. The main pyrolysis products of these resins are listed in
Section V, ENVIRONMENTAL CONTAMINATION.
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VIII. BIOLOGY
A. Absorption
The physical state of the finished formaldehyde resin products would
preclude their entry into a living system. The starting products and
the pyrolysis degradation products, however, are absorbable.
The primary candidate for absorption through the respiratory tract
would be formaldehyde itself, a flammable, colorless gas at room tem-
perature. Volkova and Sidorova (1971) found formaldehyde in the blood
of 100 workers in an amino resins manufacturing plant. Eighteen hours
after leaving the contaminated area, no formaldehyde was detected in the
blood of these workers.
Although no cases of dust inhalation with sequela by workers are
reported in the literature collected, this occurrance is a distinct
possibility. Six months after a single inhalation of the dust of
acetal resins, morphological changes were apparent in the lungs of
experimental rats (Kochetkova, Vasil'eva, Promyslova, and Sergeev,
1971).
Carbon monoxide and carbon dioxide, both toxic, are the ultimate
pyrolysis products of all plastics. The urea resins and the melamine
resins give off ammonia as a degradation product. Since these three
products are gases, they can be absorbed through the respiratory tract.
Accidental ingestion of formaldehyde resins would be unlikely.
The Food and Drug Administration (Anon., 1964) has approved the use of
melamine-formaldehyde resins for use in dinnerware. Since these resins
are hard, mar-resistant, and resistant to oils, solvents, and
greases, contamination of food from contact with these resins is most
U52
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improbable. Phenol-formaldehyde resins have been approved for use as a
food-contact surface of molded articles intended for repeated use in
contact with nonacid foods of pH above 5.0 (Anon., 1966).
8* EXCRETION/ELMINATION
Urea-formaldehyde resin and phenol-formaldehyde resin were excreted
without effect, following the administration of 5 gm/kg into the diges-
tive tracts of rabbits and rats (Galibin, 1963).
No acetal resin particles were found in the lungs of rats which
had been subjected to resin-dust inhalation for twenty-nine days. It
was assumed that the dust particles were caught in the mucous of the
respiratory tract and were excreted when the mucous was discharged
(Kopecny, Cerny, and Ambroz, 1968) .
C. TRANSPORT AND DISTRIBUTION
Kopecny, Cerny, and Ambroz (1968) carried out toxicity studies on
rats by administering the dust of acetal resins by inhalation, by
subcutaneously injections, and by intraperitoneal injections. These
authors did no transport and distribution studies, but stated that
this work should be done, in reference to organs of deposition of the
acetal powder following its injection into living systems.
D. METABOLISM AND METABOLIC EFFECTS
No metabolic studies were reported in the literature collected.
However, the granuloma formation consequent to the injection of certain
formaldehyde resins into experimental animals would indicate that these
resins are not metabolized, but are phagocytized. Granulomas were
found in the lungs of rats which had inhaled the dust of acetal resins
(Kochetkova, Vasil'eva, Promyslova, and Sergeev, 1971) and granulomas
U53
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were found in the subcutaneous tissue and in the peritoneal region of
rats after acetal resin powder had been injected at these sites
(Kopecny, Cerny, and Ambroz, 1968).
IX. ENVIRONMENTAL TRANSPORT AND FATE
A. PERSISTENCE AND/OR DEGRADATION
Finished products constructed of formaldehyde resins persevere in
the environment. They are flame-resistant, resistant to most chemicals,
and resistant to wear. Therefore, they are not subject to degradation
under normal, use-related conditions.
However, when the right conditions are present, these resins will
undergo combustion, as will all organic materials. The res.ultant com-
bustion products are listed in Section V, ENVIRONMENTAL CONTAMINATION.
B. ENVIRONMENTAL TRANSPORT
The tremendous utility of the formaldehyde resins would indicate
the degree to which materials constructed from these resins are en-
countered daily.
Environmental concern, however, should be directed to the transport
of the starting products, to the dusts formed during manufacturing,
and to the decomposition products which are released on combustion of
these resins. These subjects are discussed in Section V, ENVIRONMENTAL
CONTAMINATION.
C. BIOACCUMULATION,
Slensky and Horn (1971) studied a group of twenty-four workers
who had been exposed to the dust released during the processing of 40%
phenol-formaldehyde resin with 60% glass fiber. Some subjective
-------
signs of the effect of this substance on the skin and respiratory mucosa
were noted, but without signs of actual damage. These authors concluded
that the short time-span of exposure of the workers (4-12 months) pre-
cluded definitive results, and stated that further work must be done.
The incidence of granuloma formation in experimental animals
would indicate bioaccumulation of dusts and powders of formaldehyde
resins. Granulomas were found in the lungs of rats who had been
subjected to the inhalation of the dust of acetal resins. Granulomas
were also found in the subcutaneous tissue and in the peritoneal
region of rats after acetal powders had been injected at these cites
(Kochetkova, Vasil'eva, Promyslova, and Sergeev, 1971; Kopecny, Cerny,
and Ambroz, 1968).
X. TOXICITY
A. HUMAN TOXICITY
Every individual has daily contact with the myriad of products
which are manufactured from the formaldehyde resins, yet there is no
instance reported in the literature collected of any physiological re-
action resulting from contact with a finished product. It is the contact
between workers and the starting products used, the vapors and dusts
formed during the manufacturing process, or those intermediate, un-
finished products which might contain free formaldehyde or phenol that
is the primary source of toxicity to humans from these resins.
Formaldehyde, a colorless gas which is intensely irritating to
the mucosa and is a sensitizing agent, can be inhaled or can contact
the skin directly in either the gaseous or the dissolved states. Phenol
is a compound which is highly corrosive to the skin, and which, in
U55
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concentrated form, produces severe skin burns on contact. Urea and
melamine are not toxic and the dermatitis encountered in the manufacture
of resins containing these compounds is probably due to formaldehyde
or to other components.
1. Inhalation
Formaldehyde was detected in the blood of 100 workers who were
engaged in the manufacture of urea-formaldehyde resins. There was a
direct correlation between the concentration of formaldehyde in the
air and its level in the blood of the workers. The formaldehyde was
detectable in the blood within fifteen to seventy minutes after the
work began, and disappeared from the blood within eighteen hours after
the workers left the contaminated areas. No toxic effects from this
inhalation were reported (Volkova and Sidorova, 1971).
An increased incidence of illnesses was found among 103 workers
in a plant producing asbestos-filled phenol-formaldehyde reisin, in
comparison with the incidence of illnesses among workers engaged in
another type of production. The most common complaints were chest
pains, headaches, and skin rashes. Chronic rhinitis was found in
fourteen workers and pneumonia was discovered in three workers. These
persons had been exposed for seven years to the vapors of phenol and
of formaldehyde and to dust concentrations up to 132 mg/m3 (Troitskii,
Kuz'minykh, Andreeva, and Bunimovich, 1970).
2. Skin Contact
Dueva (1966) established a direct relationship between the sensi-
tizing effects of urea-formaldehyde resins on the skin of workers and
the amount of residual free formaldehyde contained in the resins .
Contact eczema attributable to melamine resin which had been In-
corporated into surgical bandages affected four nurses who had handled
from two to fifteen bandages daily for a period of two weeks to five
-------
months. One patient on whom this type of bandage had been applied
also showed symptoms of eczema. After the skin eruptions had healed,
cutaneous tests showed sensitivity to melamine resin, but no sensi-
tivity to formaldehyde itself (Loechel, Lenz, and Herter, 1971).
Severe, vesicular, and exudative dermatitis occurred in six pa-
tients after receiving orthopedic casts which were reinforced with
melamine-formaldehyde resin. The casts contained 10% of the melamine
resin (0.01 to 0.3% of free formaldehyde). Patch tests proved that
these patients were sensitive to formaldehyde (Logan and Perry, 1973).
Among forty-five workers who were exposed to bakelite powder
(phenol-formaldehyde resin), 13% showed eczematous skin lesions accom-
panied by intesne itching in the areas in contact with the bakelite
dust. The skin lesions were attributed to the resin dust in the air
settling on exposed surfaces of skin. No symptoms from the inhalation.
of this dust are reported (Spalinska, 1971) .
In a bakelite molding facility, twelve of the eighty persons
employed contracted dermatitis within a four-year period. The lesions
appeared as localized erythemotous papules. After the affected indi-
viduals were moved to work areas removed from the contaminated areas,
the lesions regressed (Bresson, Bertholon, and Girard, 1972) .
B. TOXICITY TO NON-HUMAN MAMMALS
1. Acute, Subacute, and Chronic Toxicity
The toxicity of urea-formaldehyde resin and phenol-formaldehyde
resin was studied in rabbits and rats. Administration into the diges-
tive tract of 5 gm/kg had no toxic effects. Cutaneous applications
of these resins were also without sequel .(Galibin, 1963).
U57
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Dust generated in the manufacturing of products consisting of 40%
phenol-formaldehyde resin and 60% glass fiber was injected irttratracheally
into rats (75 mg/rat). After 11 months the larger amount of the dust
had been eliminated. A negligible amount of small glass fiber fractions
were found in the lungs in pulmonary phagocytes or in agglomerates sur-
rounded by phagocytes. Inflammatory changes were seen in some bronchi.
Collagen tissue formation was not induced (Sklensky and Horn, 1971).
Twenty rats were subjected to inhalations of acetal resin dust.
The inhalations (approximately 5 gm per rat) were administered for thirty
minutes daily, six days a week, for one month. During the course of
the experiment, three animals perished: one from pneumonia, one
from hemmorhage into the myocardium, and the third from undetermined
causes.
Two months after the termination of the experiment, the rats were
sacrificed by exsanguination. Upon comparison with the controls, there
were no striking changes other than a slight increase in lymphoid tis-
sue and edema of the adventitia of the larger pulmonary vessels. The
adventitia were infiltrated by mononuclear and polymorphonuclear eosino-
phils.
An acetal resin suspension in 2 ml of saline was administered in-
traperitoneally to a group of five rats. Autopsy revealed small,
whitish, hard and smooth granulomas attached to the base of the serous
membranes, but no inflammatory changes. Microscopical resin particles
were encapsulated in the granulomatous tissue.
In a group of five rats which received a saline suspension of acetal
resin by subcutaneous injection into the area of the spinal column,
-------
only a single resorbing granuloma was found.
No other changes were observed in the organs of the experimental
animals—kidneys, spleen, liver, or myocardium (Kopecny, Cerny, and
Ambroz, 1968).
Kochetkova, Vasil'eva, Promyslova, and Sergeev (1971) found
phagocytes, granulomas, and inflammation in the lungs of rats of
which had inhaled acetal resin dust. Morphological changes were
apparent in the lungs six months after a single inhalation.
2. Sensitization
None of the animal studies in the literature collected reported
sensitization reactions to the formaldehyde resins.
3. Te ra to geni ci ty
No teratological effects from the formaldehyde resins is
reported in the literature collected.
4. Carcinogenicity
No malignant growths attributable to the formaldehyde resins or
to their constituents are reported in the collected literature.
However, granuloma formation was induced in the lungs of rats
which had been subjected to the inhalation of acetal resin dusts and
granulomas were found in the peritoneal and subcutaneous areas of
experimental animals at the sites of injections of saline suspensions
of acetal resin dusts (Kochetkova, Vasil'eva, Promyslova, and Sergeev,
1971; Kopecny, Cerny, and Ambroz, 1968).
5. Mutagenicity
No studies relating the formaldehyde resins to mutagenicity
are reported in the available literature.
-------
6. Behavioral Effects
There are no reports in the collected literature conceirning any
behavioral effects of the formaldehyde resins.
C. TOXICITY TO LOWER ANIMALS
No studies were encountered concerning the toxicity of the formal-
dehyde resins toward lower animals.
D. TOXICITY TO PLANTS
No reports were found reporting the toxicity of formaldehyde
resins toward plants.
E. TOXICITY TO MICROORGANISMS
Formaldehyde has had widespread use as a tissue preservative and
as a disinfectant. Phenol is a known bacteriocidal agent and, in fact,
is used as a standard of comparison in measuring the effectiveness of
other antiseptics.
Although no studies appear in the collected literature concerning
the toxicity of the formaldehyde resins to microorganisms, any resi-
dual free formaldehyde or phenol present in the resins would have a
bacteriocidal effect.
U60
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XI. CURRENT REGULATIONS
A. Food and Drug Administration
According to the Food and Drug Administration (Anon., 1964),
formaldehyde resins may be safely used as a food-contact surface on molded
articles which are used in producing, manufacturing, packing, processing,
preparing, treating, packaging, transporting, or holding food. For these
purposes, melamine-formaldehyde resins are defined as the reaction pro-
ducts of one mole of melamine and not more than three moles of formalde-
hyde in aqueous solution. The molded melamine-formaldehyde articles in
the finished form (in which they are to contact food) must not yield
chloroform-soluble extracts in excess of 0.5 mg/sq. in. of food-contact
surface.
Phenol-formaldehyde resins may also be used under the Federal Food,
Drug, and Cosmetic Act as the food-contact surface of molded articles
(Anon., 1966). This rule applies to repeated contact with nonacid food
(pH > 5.0), if the finished article meets the following specifications:
when extracted with distilled water at reflux temperatures for two hours,
using a volume-surface ratio of 2 ml of water to 1 sq. in. of surface,
the total extractives should not exceed 0.15 mg/sq. in.; the maximum
phenol detection is 0.005 mg/sq. inc., with no extracted aniline. These
determinations should be run by a spectrophotometric method sensitive to
0.006 mg/sq. in.
B. The Occupational Safety and Health Act
The Occupational Safety and Health Act of April 28, 1971, has
special significance for the plastics industry, since this industry has
such a poor safety record. The act states that each employer "shall
U61
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furnish to each of his employees, employment and a place of employment,
which are free from recognized hazards that are causing, or are likely
to cause, death or serious physiological harm to his employees".
This bad safety record is a matter of accidents with the equipment,
however, and not a matter of hazard from contaminated areas (Anon., 1971; 2).
Other OSHA regulations are discussed in Section XII, STANDARDS.
C. Department of Transportation
The Department of Transportation has issued flammability rules for
automotive interiors and has proposed upgrading regulations covering
passenger airplane furnishings (Anon., 1972; 2). The formaldehyde resins
would be included in the plastics which would be under consideration here.
Test methods for special materials for aircraft applications will be
established by ASTM Committee F-7. Tests will be developed to evaluate
safe performance of cleaners for plastics and other exterior and interior
surfaces (Anon., 1973).
D. Air and Water Acts
Acetal copolymer has been approved by the National Science Foundation
for use in contact with water to be used for drinking (Modern Plastics
Encyclopedia, 1970-71, pg. 88) .
E. State, Federal, and Foreign Regulations
A New York City tax on plastic containers was declared unconstitutional
on November 11, 1971. Justice Saul S. Streit of the Supreme Court of New
York State ruled that such discrimination against plastics was arbitrary
and unreasonable, and thus a violation of the equal protection clauses
of the Federal and New York State Constitutions. Justice Streit stated,
"There was not one shred of evidence presented herein which demonstrates
that any form of container, glass, metal, or paperboard, is any more
-------
recyclable than plastic containers .... It thus appears that the dis-
crimination against plastic containers does not rest on any reasonable
basis in relation to the objective of promoting the recycling of con-
tainers, plastics or otherwise" (Kestler, 1971; Anon., 1972; 3).
Some further regulations are discussed in Section XII, STANDARDS.
XII. STANDARDS
A. Threshold Limit Values
In June, 1972, a federal regulation was published under the Occupa-
tional Safety and Health Act (OSHA) , limiting the concentration of as-
bestos fibers (a filler for formaldehyde resins) in work areas to 5
units per cc of air, which is to be reduced to 2 units per cc of air by
July 1, 1976. The previous threshold limit value for asbestos, published
by the American Conference of Government Industrial Hygienists (ACGIH)
was a limit of 12 units per cc of air.
To obviate investments in protective clothing, improved dust-
collection and ventilation systems, and periodic examinations of workers,
General Electric has complied with OSHA's ruling by changing its phenolic
compound formulation. The identity of the substitute filler has not been
revealed, but GE has said that it has been cleared by the Food and Drug
Administration (Anon., 1972; 1).
The following figures show the maximum permitted atmospheric con-
centrations of formaldehyde and of phenol (in ppm and mg/m3) in factories
in Great Britain, the United States, and Russia.
U63
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COUNTRY FORMALDEHYDE PHENOL
ppm mg/m3 ppm mg/m3
Great Britain - 12 - -
United States 56 5 19
U.S.S.R. - 1 5
B. Flammability
The National Materials Advisory Board, which evaluates materials on
request from federal agencies, has begun a three-year study to gather,
correlate, and analyze material on fire testing and fire behavior of
plastics. The ultimate goal is to develop a model test that will dupli-
cate any fire-situation performance. A twenty-member committee on the
Fire Safety Aspects of Polymeric Materials has been formed.
The Fire Retardant Chemicals Association, formed in 1973, has launched
an organized inquiry into the fire safety of polymerics. The aims of this
new group include better communication with government agencies, and inter-
action with industry groups (Macbride, 1973).
The Underwriters' Laboratories are developing systems for plastic
product safety. The UL temperature index (Reymers, 1970; a) correlates
numerically with the temperature rating (the maximum temperature in °C
above which a material may degrade prematurely and, therefore, be unsafe).
The formaldehyde resins have been given the following temperature indices:
Urea-formaldehyde resins 100°C
Molded melamine resins (excluding fiber-reinforced) 130°C
Molded phenolic resins (excluding fiber-reinforced) 150°C
These figures apply to heat and pressure molded resins only; they do
not apply to those resins intended for casting or pouring (Reymers, 1970; a).
-------
Among the Underwriters' Laboratories flammability indexing tests are
the self-extinguishing tests (the phenol-formaldehyde resins hold a Group
I rating), the slow-burning tests, the hot-wire ignition tests, the high-
current-arc ignition tests, the high-voltage-arc ignition tests, and the
high-voltage-arc track tests.
Prior to 1973, the classifications were defined as follows:
SE-0 "Self-extinguishing, Group 0" Do not release flaming
particles and do not con-
tinue to flame longer than
10 seconds.
SE-1 Group I Do not release flaming
particles or drops.
SE-2 Group II Release flaming particles
or drops which burn only
briefly.
(Reymers, 1970; b)
In September, 1973, Underwriters' Laboratories distributed a revised
edition of its UL-94 test for flammability of plastics materials. The
terms "SE" (self-extinguishing) and "SB" (slow-burning) have been deleted.
"SE" designations have been replaced by the letter code "VE" (vertical
flame extinguishing tests) and "SB" designations have been replaced by
"HB" (horizontal burn tests). The testing parameters remain unchanged.
The American Society for Testing and Materials is in the process of
revising the terminology of its D-635 flammability test. All references
to the "NB" classification (nonburning by this test) are eliminated. The
text contains a warning that the indicated properties are based on small-
scale laboratory tests, and in no case are to be used as an assessment of
actual fire hazards (Anon., 1973; 1).
-------
LITERATURE CITED
Anon (1964), "Food Additives, Melamine - Formaldehyde Resins in
Molded Articles", Federal Register 29_, 6952-3, (May 27) 12249
Anon (1966), "Food Additives. Phenolic Resits in Molded Articles",
Federal Register _31, 2476-7 (Feb. 8) 15775
Anon (1970;!), "More Help for Solid-Waste Dispensability; Modern
Plastics 47^ (8), 67-8
Anon (1970;2), "Are These Incinerators the Answer to Plastics Waste?",
Modern Plastics 4J (10, 102-3
Anon (1971;!), "A Closed-Loop Approach to Industrial Plastics Wastes",
Modern Plastics 48 (7), 44-45
Anon (1971;2), "The Government is Looking at your Safety Practices.
Are you Ready?", Modern Plastics, 48 (7), 36-8
Anon (1972;!), "Phenolics Get the Lead Out, and Some of the Asbestos
Tco", Modern Plastics 49_ (12), 48-50
Anon (1972;2), "Business and Industry", Modern Plastics _49_ (6), 125-126
Anon (1972;3), "Things Are Looking Up", Modern Plastics _48 (12), 40
Anon (1973), "Industry and Market News", Modern Plastics _50_ (12), 103
Anon (1973;!), "It's a Small Start, but UL and ASTM FlammabLlity
Tests Drop the Adjectives", Modern Plastics 50; (10), 16
Ardelt, H. W.; and Opel, P. H. (1962), "Determination of Free
Formaldehyde in Urea-Formaldehyde Foams, Plaste Kautschuk 9_,
115-16 (Ger.) 11385
Braun, D,, and Jung, J. C. (1970), "Simple Qualitative Determination
of Aminoplasts", Gutnmi, Asbest, Kunstst, 23_ (6), 618, 620, 622
(Ger.) 12135
Bresson, J. R., Bertholon, J., and Girard, R. (1972), "Secondary
Dermatitis from Phenolplastic Resins", Arch. Mai. Prof., 83_,
199-201 (Fr.) ' 16776
-------
Chemical Economics Handbook, Stanford Research Institute, Menlo
Park, Calif.
Chernousova, L. N., Lok, S. M. and Filiko, T. M. (1970), "Prevention
of Occupational Dermatoses Among Adolescents Having Contact
with Phenol-Formaldehyde Resins", Gig. Sanit. 15 (7), 103-
104 (Russ.) 16406
Dotreppe-Grisard, N. (1968), "Compounds Obtained by Combustion
of Plastics", Trib. CEBEDEAV (Centre Beige Etude Doc. Eaux),
21 (294), 274-8 Fr. 12072
Dueva, L. A. (1966), "Experimental Materials Contributing to the
Establishment of Hygienic Standards for Urea-Formaldehyde
Resins", Gig. Tr. Prof. Zabol. 10 (11), 39-43 (Russ.) 12475
Galibin, G. P. (1963), "Action of Hardened Synthetic Resins en
Animal Systems", Toksikol - Novykh Prom. Khim. Veshehestv 5_,
45-50 (Russ.) 14196
Hauck, J. E. (1971;!), "Engineering and Processing News", Modern
Plastics 48 (7), 72-76
Hauck, J. E. (1971;2), "Plastiscope 2", Modern Plastics 4jJ (5),
86-88
Hiramatsu, K. (1967), "Mass-Spectrometric Analysis of Pyrolysis
Products of Melamine Formaldehyde and Urea Formaldehyde Resins",
Osaka Juritsu Kogyo-Shoreikan Hokoku, 43, 28-33 (Jap.) 12309
Hirt, R. C., King, F. T., and Schmitt, R. G. (1954), "Detection
and Estimation of Melamine in Wet-Strength Paper by Ultraviolet
Spectrophotometry", Anal. Chem. 26^ (8), 1273-4 12264
Karapetyan, S. 0. (1971), "Diseases of the Nervous System in Workers
of the Echmiadzinski Plastics Factory", Zh. Ekop. Klin. Ed.
11 (2), 100-103 (Russ.) 16437
Kestler, J. (1971), "What Are the Implications of the Overturning
of New York City's Tax Law?", Modern Plastics 48^ (12), 18
Kirk-Othmer Encyclopedia of Chemical Technology (1964), 3^, 770-9,
Interscience Pub.
Kochetkova, T. A., Vasil'eva, 0. I., Promyslova, A. D., and Sergeev,
A. N. (1971), "Assessment of Morphological Changes in the Lungs
of Experimental Animals Caused by the Dust of Some Powdered
Polymers", Gig. Sanit. 36 (7), 75-8 15393
1467
-------
Kopecny, J., Cerny, E., and Ambroz, D. (1968), "The Effect of
Polyformaldehyde Dust in Rat Tissues", Scr. Med. ^L (7-8),
405-9 12109
Loechel, I., Lenz, U. and Herter, A. (1971), "Contact Eczema from
a Plastic-Containing Plaster Bandage", Deut. Gesundheitsw.
26 (11), 511-13 (Ger.) 14295
Logan and Perry (1973), "Contact Dermatitis to Resin-Containing
Casts", Clin. Orthop. 90, 150-2 16310
Macbride, R. R. (1973), "Two Outstanding Examples of the Systems
Engineering Approach, "Modern Plastics 50 (11), 64-67.
Modern Plastics (1973;7), "Plastiscope 3", 50 (7), 100
i
Modern Plastics (1974;!), "We Produced over 13 Million Tons of
Resin in '73? Well, Where is it? And How about '74?",
51 (1), 36-47
Modern Plastics Encyclopedia (1970-71), V7 (10 A), pg. 88,
October 1970, McGraw-Hill Inc.
Modern Plastics Encyclopedia (1973-74), 5<3 (10 A), October 1973,
McGraw-Hill Inc.
Reymers, H. (1970;a), Modern Plastics 47 (9), 78-81
Reymers, H. (1970;b), Modern Plastics, _47_ (10), 92-8
Sklensky, B. and Horn, V. (1971), "The Effect of AG 4(Phenolformaldehyde
Resin with Glass Fiber) upon Rat Lung", Pracovni Lekar (Prague)
23 (7): 230-232 (Czech.) 16132
Spalinska, L. (1971), "Skin Lesions in Workers Manufacturing Goods
from Bakelite Powder", Przeg. Derm. .58^ (5), 555-60 (Pol.) 16215
Stasenkova, K. P. and Mellnikova, R. N. (1961), "Toxicity of Certain
Iso Alcohols, Higher Alcohols and Melamine-Formaldehyde Resins",
Toksikol. Novykh Prom. Khim. Veschchestv _3, 108-12 (Russ.) 11386
Stevens, M. P., and Percival, D. F. (1964), "Gas Chromatographic
Determination of Free Phenol and Free Formaldehyde in Phenolic
Resins", Anal. Chem. 36 (6), 1023-4 14063
U68
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Surkova, F. M. and Saperova, E. F. (1970), "The Change in the Serum
Proteins in Persons in Contact with Epoxide and Phenol-
Formaldehyde Resins", Tr. Persn. Med. Inst. 82, 112-114 16469
Swann, M. H., and Weil, D. J. (1956), "Colorimetric Determination
of Phenoloic Resins", Anal. Chem. 28, 1463-5 10700
Troitskii, S. Yu., Kuz'minykh, A. N., Andreeva, T. D., and Burnimovich,
G. I. (1970), "Hygienic Features of Working Conditions Prevailing
during Production of Phenoplasts with Asbestos Filling Material",
Gig. Sanit. 35_ (9), 89-91 (Russ.) 14990
Vankos, J., Borza, L., and Palfi, B. A. (1967), "Formaldehyde-Textile
Dermatitis", Z. Haut-Geschlechtskr. 42^ (5), 147-52 (Ger.) 12915
Volkova, Z. A. and Sidorva, E. A. (1971), "Blood Formaldehyde Content
in Persons Exposed to the Effect of Urea-Formaldehyde Resins",
Gig. Tr. Prof. Zabol _15 (5), 44-46 (Russ.) 16474
Wise, J. K. and Smith, C. D. (1967), "Infrared Spectrometric
Examination of Paper. II. Determination of Urea-Formaldehyde
Resin", Anal. Chem. 39 (14) 1702-5 12451
Zulaica, J., and Guiochon, G. (1966), "Identification of Phenol-
Formaldehyde Resins by Gas-Chromatographic Analysis of Their
Pyrolysis Products", J. Polymer Sci., Pt. B 4 (8), 567-71 11063
-------
o-NITROCHLOROBENZENE
SUMMARY AND CONCLUSION
Since the only use for the material seems to be for conversion to
dinitrochlorobenzene, it is unlikely that any more is released to the
environment than that which comes from waste waters of production and
conversion plants. This is fortunate because the limited data available
indicate very high toxicity and very low biodegradability. Partial
metabolism appears only to convert it to other toxic materials such as
chloroaniline. Assuming that proper safeguards are observed In the in-
dustries involved, the only likely hazard is to life in and dependent
upon bodies of water receiving the industrial waste, especially, in view
of the density and water insolubility, bottom dwellers.
Production figures for o-nitrochlorobenzene were not published
after 1967, but the 1963-1967 period was one of rapid increase. If the
proportion of sales to production was maintained after 1967, then pro-
duction continued to rise through 1969 (the last year for which sales
figures are available). As of 1972 imports were not significant.
-------
0-NITROCHLOROBENZENE
I. PHYSICAL PROPERTIES
This compound is a crystalline solid having (°C): mp 32-3, bp 245-6,
flash point 127, d 1.368(22/4); it is soluble in benzene, ethanol, and
ether, insoluble in water (0.44 g/1 at 20°).
II. PRODUCTION
Table I. Production-Importation of o-Nitrochlorobenzene
Year
1963
1964
1965
1966
1967
1968
1969
1970
1971
1972
Made in USA ' ' Imported into USA
8,570 (1,779)
9,118 ( )
12,841 ( )
16,443 ( )
15,535
—
—
—
0.12
56
a - units are metric tons
b - figure in ( ) is for a mixture of o- and p- isomers
c - amounts sold were: 4,864; 4,379; 4,782; 5,590; 5,629; 6,637; 9,255;
__j ~~l ~~
Currently the only known domestic producers are American Aniline Products,
duPont, and Monsanto. There is no indication that the o/p mixture has
been produced since 1966.
kll
-------
III. USES
The only known use is as an intermediate for dye synthesis, being
converted to 2,4-dinitrochlorobenzene.
IV. CURRENT PRACTICE
Sax indicates that the IATA regulations for liquid nitrochloroben-
zene require the container to bear a poison label, also the Poison B
label; quantities are limited to one liter on a passenger craft, 220
liters on a cargo plane.
No information bearing on disposal methods was found.
V. ENVIRONMENTAL CONTAMINATION
No references were found which indicated the occurrence of this com-
pound in the environment.
VI. MONITORING AND ANALYSIS
Piotrowski (1965) reported a colorimetric method for measuring
chloronitrobenzenes in air. Dyatlovitskaya and Potemkina (1963) re-
ported a colorimetric/polarographic method for distinguishing between
chloronitrobenzenes and nitrobenzene present together. Fleszar (1964)
reported an extraction/polarographic method for measuring 5-50 ppm of
nitrochlorobenzenes in water. Kolbasov et al (1962) discussed an infra-
red technique for measuring the various nitrochloro-, 2,5-dichloronitro-,
and 3,5-dichloronitrobenzenes present in a mixture. Stanescu and Radules-
cu (1970) reported an infrared method for analyzing a mixture of the ortho
and para isomers ranging from 4:1-1:4.
Hashimoto et al (1965) determined R values in five solvents using
thin layer chromatography (TLC). Obruba and Navratil (1967) used TLC to
separate nitration mixtures of chlorobenzene consisting of the ortho and
para mononitros, di- and trinitros.
1*72
-------
Gas liquid chromatography is the favorite analytical tool for
nitrochlorobenzenes and has been discussed in the following papers:
Bykova et al (1969), dimethylsiloxane on diatomite, katharometer;
Habboush and Norman (1962), dinonyl phthalate, tritolyl phosphate,
trinitrofluorenone on Embacel, flame ionization detector; Habboush
and Tameesh (1970), polyethyleneglycol 1500, polyethyleneglycol succi-
nate on Chromosorb P, thermal conductivity detector; Nemova et al (1969),
polyethyleneglycol 1000 on INZ-500; Roseira (1970), involved initial
conversion to nitrobutylbenzene; Stolyarova et al (1965), polyethylene-
glycol 1000 on brick;; Volkova et al (1972), polyethyleneglycol 1500 on
Celite 545, flame ionization detector; Zielinski et al (1967), tri-
fluoropropylmethyl silicone on Chromosorb G, electron capture detector.
VII. CHEMICAL REACTIVITY
No information bearing on this matter was found.
VIII. BIOLOGY
A. Metabolic Effects
Bray et al (1956) compared the metabolism of the three nitrochloro-
benzene isomers in rabbits. The dose of the ortho isomer given was a
single one of 0.1 g/kg, and was the largest which elicited no signs of
toxicity; in comparison, 0.2 g/kg doses of the other two isomers were
still non-toxic. Urine and feces were analyzed for up to 48 hours after
administration of the dose, by which time no more metabolites were
present.
It was possible to account for 82% of the dose: 42% in the urine
as a glucuronide; 24% in the urine as a sulfate; 9% in the urine and 0.3%
in the feces as free o-chloroaniline; 7% in the urine as a mercapturate
U73
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(the values for the 42, 24, and 7% conjugates are medians of three
animals; the urinary free chloroaniline was a pooled value of six
animals).
The following free phenols (totalling 5% of the dose) were found in
the urine in substantial quantities: 3-amino-4-chloro-, 4-amino-3-chloro-,
3-chloro-4-nitro-, and in trace quantities: 2-chloro-3-nitro~, 4-chloro-
3-nitro-, 3-chloro-2-nitro-, and 3-amino-2-chloro-. The mercapturate
found was the 2-nitro-phenyl. The following phenols were present as the
glucuronide or sulfate conjugates in substantial quantities: 3-chloro-4-
nitro-, 3-amino-4~chloro-, and 4-amino~3-chloro-, and in trace quantities:
2-chloro-3-nitro-, 2-amino-3-chloro-, and 3-amino-2-chloro-.
B. Physiological Effects
Shirai (1953) reported that factory workers exposed to riitrochloro-
benzene had higher blood glutathione levels than normal people even when
not showing any toxic symptoms; seasonal variation of this level was nor-
mal in the workers.
Frenkel and Gordienko (1960) found that by poisoning rats with nitro-
chlorobenzene the following changes in brain tissue chemistry occurred:
preformed ammonia and glutamine levels increased; protein amino N fell
by 15 mg%; none in levels of nonprotein N, glycogen, or ATP.,
Frenkel (1963) reported results similar to those of the 1960 paper,
adding that creatine phosphate level increased, and hypothesizing that
these changes result from inhibition of anabolic synthetic reactions.
IX. ENVIRONMENTAL EFFECTS
A. Persistence and/or Degradation
Ludzack and Ettinger (1963) found that no more than 20% of the
U7U
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theoretical amount of carbon dioxide was eventually produced from the
degradation of o-nitrochlorobenzene in a sample of river water over an
80 day test period. In comparison, 50% of the theoretical amount of
C(>2 from acetophenone evolved in five days.
Alexander and Lustigman (1966) found that there was no significant
splitting apart of the ring by soil micro-organisms in a 64 day test
period, indicating that the chemical was certainly not a food source for
them. Determination of lesser degrees of metabolism or degradation was
beyond the scope of their experiment.
B. Environmental Transport
C. Bioaccumulation
No information was found on either of these considerations.
X. TOXICITY
A. Human
Lutowiecki (1960) reported on a test for skin sensitivity in new
workers, but the results are difficult to obtain.
Sax reported that acute or chronic local toxicity was unknown, but
acute or chronic systemic toxicity from either ingestion or inhalation
was high. The pathology was methemoglobin formation, cyanosis, and
other blood changes; the effects were analogous with nitrobenzene, and
were cumulative. Industrially, contact is most likely to be made from
dust.
B. Birds and Animals
Bray et al (1956) reported that a single dose to rabbits of over
0.1 g/kg was toxic enough to interfere with their metabolism study (see
VIII, A.), but didn't further elaborate.
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Navrotskii (1953) reported that the production of methemoglobin
following nitrochlorobenzene poisoning in animals was not affected by
administration of adrenaline, but was initially depressed by acetylcho-
line, and blocked during the sleep induced by chloral hydrate or Medinal.
When the narcotic effect of the latter two ceased and the animal awoke,
methemoglobin production commenced.
Rusakov et al (1973) found that 8 yg/cu m was the minimum atmos-
pheric concentration which would elicit allergenic sensitization in rats
and guinea pigs. Transfer of serum from sensitized rats to guinea pigs
resulted in allergic symptoms in the latter.
C. Lower Animals
No information on toxicity to this class of life was fouiid.
D. Plants
Eckert (1962) found that 50% reductions in growth of cu'cumber and
mung bean seedlings resulted from contact with 115 and 190 yM solutions,
respectively, for six days.
E. Micro-organisms
Eckert (1962) found that 50% reductions in the propagation of the
soil fungi Rhizoctonia solani and Pythium ultimum resulted from contact
with 310 and 1,000 yM solutions, respectively.
Richardson (1968) found that the vapor phase toxicity: to P. ulti-
mum was lower for the nitrochloro below 250 ppm (the compound being
dispersed in soil at various concentrations, and the fungi being sus-
pended above the soil); to R. solani was about the same as for nitroben-
zene; to the saprophytic fungus Trichoderma viride was a bit lower than
nitrobenzene.
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Romanova and Rapoport (1971) reported a reduced viability of spores
of Actinomyces sphaeroides after two-hour suspension in millimolar
nitrochlorobenzene.
XI. CURRENT REGULATIONS
XII. STANDARDS
No information was found other than the IATA requirement mentioned
in Section IV of this report.
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LITERATURE CITED
Alexander, M. and B. K. Lustigman (1966). Effect of Chemical Structure
on Microbial Degradation of Substituted Benzenes. J. Agr. Food Chem.
14(4), 410-13 10642
Bray, H. G., S. P. James, and W. V. Thorpe (1956). Metabolism of the
Monochloronitrobenzenes in the Rabbit. Biochem. J. 64, 38-44 10696
Bykova, L. I., L. Ya. Gertsberg, and L. K. Etnets (1969). Determination
of Unsubstituted Aromatic Compounds and their Nitro Derivatives. Khim.
Prom. Ukr. 1969, (4), 43-4 15679
Chemical Week Buyers Guide - 1974
Dyatlovitskaya, F. G. and S. K. Potemkina (1963). Determination of Nitro-
benzene in Industrial Wastes by Using Indophenol and Polarographic Methods.
Gigiena i Sanit. 28, No.l, 38-44 11718
Eckert, J. W. (1962). Fungistatic and Phytotoxic Properties of Some
Derivatives of Nitrobenzene. Phytopathology 52, 642-9 11379
Fleszar, B. (1964). Determination of Trace Amounts of Nitrochlorobenzenes
in Water. Chem. Anal. (Warsaw) 9(6), 1075-82 14660
Frenkel, S. R. (1963). Metabolic Studies in Pathogenesis Due to Toxic
Substances. Gigiena i Fiziol. Truda, Proizv. Toksikol., Klinika Prof.
Zabolevanii (Kiev: Cos. Izd. Med. Lit. Ukr. SSR) Sb. 1963, 93-6 12417
Frenkel, S. R. and E. A. Gordienko (1960). Changes in Brain Metabolism
During Intoxication. Prom. Toksikol., Moscow, Sbornik 1960, 32-41 10548
Habboush, A. E. and R. 0. C. Norman (1962). Analysis of Mixtures of
Isomeric Benzenoid Compounds by Gas-Liquid Chromatography. J. Chromatogr.
7, 438-46 12614
Habboush, A. E. and A. H. Tameesh (1970). Gas-liquid Chromatography
of Disubstituted Benzene Isomers. II. Separation and Study of the
Halonitrobenzenes, Anisoles, and Toluenes. J. Chromatogr. 53(2), 151-
62 15847
Hashimoto, S., J. Sunamoto, and I. Shinkai (1965). Thin Layer Chroma-
tography of Aromatic Nitro Compounds and Corresponding And.no Compounds.
Kogyo Kagaku Zasshi 68(12), 2510-11 11248
I
Kirk-Othmer, Encyclopedia of Chemical Technology, 2nd ed.,, Vol. 7,
Wiley, N. Y. (1965)
Kolbasov, V. I., S. B. Bardenshtein, R. V. Dzhagatspanyan, and E. V.
Zakharov (1962). Infrared Analysis of Technical m-Chloro-nitrobenzene.
Zavodsk. Lab. 28, 1326-7 12582
Ludzack, F. J. and M. B. Ettinger (1963). Biodegradability of Organic
Chemicals Isolated from Rivers. Purdue Univ., Eng. Bull., Ext. Ser.
No. 115, 278-82 14203
U78
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Lutowiecki, J. (1960). Prophylaxis of Occupational Dermatosis in the
Chemical Industry. Symp. Dermatol., Corpus Lection. Univ. Carolina,
Prague 3, 79-82 (Pub. 1962) 14120
Navrotskii, V. K, (1953). The Area of Industrial Toxicology as an As-
pect of Pavlov Neurology. Gigiena i Sanit. 1953, No. 4, 3-11 15036
Nemova, M. M., V. N. Pertsova, and S. L. Dobychin (1969). Gas-chromatogra-
phic Analysis of Chloronitrobenzenes. Tr., Cos. Inst. Prikl. Khim.
1969, No. 62, 173-5 14991
Obruba, K. and F. Navratil (1967). Chromatographic Identification and
Determination of Chlorobenzene Nitration Products. Chem. Prum. 17(3),
155-7 12909
Piotrowski, J. (1965). Colorimetrlc Determination of Nitrobenzene and
its Chloro Derivatives in Mr. Chem. Anal. (Warsaw) 10(1), 55-65 15871
Richardson, L. T. (1968). Selective Vapor Activity of Chloronitro- and
Chlorobenzenes in Soil. Phytopathology 58(3), 316-22 12361
Romanova, N. B. and I. A. Rapoport (1969). Mutagenic Model of a Study
of Nitro Compounds as Protective Agents from Ultraviolet Radiation.
Teor. Khim. Mutageneza, Mater. Vses. Soveshch., 4th, 7-10 (Pub. 1971)
11192
Roseira, A. N. (1970). Gas-phase Chromatographic Behavior of Nitrobu-
tylanilines. Identification of Nitrochlorobenzenes. An. Acad. Brasil.
Cienc. 42(4), 711-15 11106
Rusakov, N. V., G. I. Korotkova, and V. Sh. Bikbulatov (1973). Experi-
mental Study of the Allergenic Action of Ortho- and Paranitroehloroben-
zene. Gigiena 1 Sanit., No. 3, 13-16 16456
Sax, N. I. Dangerous Properties of Industrial Materials, 3rd ed.,
Reinhold, N. Y. (1968)
Shirai, A. (1953). Quantity of Reduced Glutathione in the Blood of
Workers in Toxic Environments. Nisshin Igaku 40, 20-4 13508
Stanescu, G. and 0. Radulescu (1970). Ir Spectrophotometric Method for
Determining Mixtures of o- and p-Nitrochlorobenzene. Rev. Chim.
(Bucharest) 21(2), 70 15533
Stolyarova, G. N., V. N. Pertsova, T. K. Lozinskaya, M. M. Nemova, and
S. L. Dobychin (1965). Chromatographic Analysis of Isomers of Nitro-
chlorobenzene. Gaz. Khromatogr., Moscow No. 3, 94-6 12903
United States Tariff Commission Report, Synthetic Organic Chemicals
Volkova, G. G., Yu. A. Galishevskii, and A. G. Klein (1972). Gas-
chromatographlc Analysis of Technical 2,4-Dinitrochlorobenzene. Zavod.
Lab. 38(2), 156-7 11131
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Weast, R. C., ed., Handbook of Chemistry and Physics, 52nd ed., Chemical
Rubber Co., Cleveland (1971)
Zielinski, Jr. W. L., L. Fishbein, and R. 0. Thomas (1967). Relation
of Structure to Sensitivity in Electron Capture Analysis. III. Chloro-
nitrobenzenes, Anilines, and Related Derivatives. J. Chromatogr.
30(1), 77-85 13168
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 REPORT NO.
EPA-560/2-76-005
4. TITLE AND SUBTITLE
2,
3. RECIPIENT'S ACCESSION NO.
A Literature Survey Oriented Towards
Adverse Environmental Effects Resultant from the Use
of Azo Compounds, Brominated Hydrocarbons, EDTA,
Formaldehyde Resins, and O-Nitrochlorobenzene
5. REPORT DATE
June 1976
6. PERFORMING ORGANIZATION CODE
7 AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO
Richard Mason
Sh:rley C. Sweeney
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Science Information Services Department
Franklin Institute Research Laboratories
Philadelphia, Penn. 19103
10. PROGRAM ELEMENT NO.
2LA328
11. CONTRACT/GRANT NO.
Contract No. 68-01-2212
12 SI ONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
Office of Toxic Substances
Washington, D.C'. 20460
13. TYPE OF REPORT AND PERIOD COVt >
Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A review of the literature published from 1953 through 1973 was conducted to
prepare this report on the physical and chemical properties of azo compounds,
brominated hydrocarbons, EDTA, formaldehyde resins and o-nitrochlorobenzene, on
environmental exposure factors related their consumption and use, on the health a
environmental effects resulting from exposure to these substances and on any
applicable regulations and standards governing their use.
i1
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
! azo compounds
brominated hydrocarbons
EDTA
formaldehyde resins
o-nitrochlorobenzene
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Grcmp
-chemical properties
-physical properties
-environmental effects
-environmental exposure
06/F,0,P,T
07/A,C,D
13. DISTRIBUTION STATEMENT
Release unlimited
19. SECURITY CLASS (ThisReport)
unclassified
21. NO. OF PAGES
20 SECURITY CLASS (Thispage)
unclassified
22 PRICE
EPA Form 2220-1 (9-73)
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